diff --git a/changelog/4548.removal.rst b/changelog/4548.removal.rst
new file mode 100644
index 000000000..bd47b1d51
--- /dev/null
+++ b/changelog/4548.removal.rst
@@ -0,0 +1,3 @@
+An error is now raised if the ``pytest_plugins`` variable is defined in a non-top-level ``conftest.py`` file (i.e., not residing in the ``rootdir``).
+
+See our `docs <https://docs.pytest.org/en/latest/deprecations.html#pytest-plugins-in-non-top-level-conftest-files>`__ for more information.
diff --git a/doc/en/deprecations.rst b/doc/en/deprecations.rst
index 781d05b77..ca95bab38 100644
--- a/doc/en/deprecations.rst
+++ b/doc/en/deprecations.rst
@@ -81,16 +81,6 @@ As part of a large :ref:`marker-revamp`, :meth:`_pytest.nodes.Node.get_marker` i
 :ref:`the documentation <update marker code>` on tips on how to update your code.
 
 
-pytest_plugins in non-top-level conftest files
-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
-
-.. deprecated:: 3.5
-
-Defining ``pytest_plugins`` is now deprecated in non-top-level conftest.py
-files because they will activate referenced plugins *globally*, which is surprising because for all other pytest
-features ``conftest.py`` files are only *active* for tests at or below it.
-
-
 marks in ``pytest.mark.parametrize``
 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
 
@@ -242,6 +232,16 @@ You can consult `funcarg comparison section in the docs <https://docs.pytest.org
 more information.
 
 
+pytest_plugins in non-top-level conftest files
+~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
+
+*Removed in version 4.0.*
+
+Defining ``pytest_plugins`` is now deprecated in non-top-level conftest.py
+files because they will activate referenced plugins *globally*, which is surprising because for all other pytest
+features ``conftest.py`` files are only *active* for tests at or below it.
+
+
 ``Config.warn`` and ``Node.warn``
 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
 
diff --git a/src/_pytest/config/__init__.py b/src/_pytest/config/__init__.py
index 9ad7da008..f20bb036e 100644
--- a/src/_pytest/config/__init__.py
+++ b/src/_pytest/config/__init__.py
@@ -31,6 +31,7 @@ from _pytest._code import ExceptionInfo
 from _pytest._code import filter_traceback
 from _pytest.compat import lru_cache
 from _pytest.compat import safe_str
+from _pytest.outcomes import fail
 from _pytest.outcomes import Skipped
 from _pytest.warning_types import PytestWarning
 
@@ -429,11 +430,11 @@ class PytestPluginManager(PluginManager):
                         PYTEST_PLUGINS_FROM_NON_TOP_LEVEL_CONFTEST
                     )
 
-                    warnings.warn_explicit(
-                        PYTEST_PLUGINS_FROM_NON_TOP_LEVEL_CONFTEST,
-                        category=None,
-                        filename=str(conftestpath),
-                        lineno=0,
+                    fail(
+                        PYTEST_PLUGINS_FROM_NON_TOP_LEVEL_CONFTEST.format(
+                            conftestpath, self._confcutdir
+                        ),
+                        pytrace=False,
                     )
             except Exception:
                 raise ConftestImportFailure(conftestpath, sys.exc_info())
diff --git a/src/_pytest/deprecated.py b/src/_pytest/deprecated.py
index 467c73584..02b26ba77 100644
--- a/src/_pytest/deprecated.py
+++ b/src/_pytest/deprecated.py
@@ -69,10 +69,14 @@ WARNS_EXEC = PytestDeprecationWarning(
     "See https://docs.pytest.org/en/latest/deprecations.html#raises-warns-exec"
 )
 
-PYTEST_PLUGINS_FROM_NON_TOP_LEVEL_CONFTEST = RemovedInPytest4Warning(
-    "Defining pytest_plugins in a non-top-level conftest is deprecated, "
+PYTEST_PLUGINS_FROM_NON_TOP_LEVEL_CONFTEST = (
+    "Defining 'pytest_plugins' in a non-top-level conftest is no longer supported "
     "because it affects the entire directory tree in a non-explicit way.\n"
-    "Please move it to the top level conftest file instead."
+    "  {}\n"
+    "Please move it to a top level conftest file at the rootdir:\n"
+    "  {}\n"
+    "For more information, visit:\n"
+    "  https://docs.pytest.org/en/latest/deprecations.html#pytest-plugins-in-non-top-level-conftest-files"
 )
 
 PYTEST_CONFIG_GLOBAL = PytestDeprecationWarning(
diff --git a/testing/deprecated_test.py b/testing/deprecated_test.py
index bb2c17c45..a6ef4739b 100644
--- a/testing/deprecated_test.py
+++ b/testing/deprecated_test.py
@@ -113,17 +113,15 @@ def test_pytest_plugins_in_non_top_level_conftest_deprecated(testdir):
     """
     )
     res = testdir.runpytest(SHOW_PYTEST_WARNINGS_ARG)
-    assert res.ret == 0
+    assert res.ret == 2
     msg = str(PYTEST_PLUGINS_FROM_NON_TOP_LEVEL_CONFTEST).splitlines()[0]
     res.stdout.fnmatch_lines(
-        "*subdirectory{sep}conftest.py:0: RemovedInPytest4Warning: {msg}*".format(
-            sep=os.sep, msg=msg
-        )
+        ["*{msg}*".format(msg=msg), "*subdirectory{sep}conftest.py*".format(sep=os.sep)]
     )
 
 
 @pytest.mark.parametrize("use_pyargs", [True, False])
-def test_pytest_plugins_in_non_top_level_conftest_deprecated_pyargs(
+def test_pytest_plugins_in_non_top_level_conftest_unsupported_pyargs(
     testdir, use_pyargs
 ):
     """When using --pyargs, do not emit the warning about non-top-level conftest warnings (#4039, #4044)"""
@@ -143,7 +141,7 @@ def test_pytest_plugins_in_non_top_level_conftest_deprecated_pyargs(
     args = ("--pyargs", "pkg") if use_pyargs else ()
     args += (SHOW_PYTEST_WARNINGS_ARG,)
     res = testdir.runpytest(*args)
-    assert res.ret == 0
+    assert res.ret == (0 if use_pyargs else 2)
     msg = str(PYTEST_PLUGINS_FROM_NON_TOP_LEVEL_CONFTEST).splitlines()[0]
     if use_pyargs:
         assert msg not in res.stdout.str()
@@ -151,7 +149,7 @@ def test_pytest_plugins_in_non_top_level_conftest_deprecated_pyargs(
         res.stdout.fnmatch_lines("*{msg}*".format(msg=msg))
 
 
-def test_pytest_plugins_in_non_top_level_conftest_deprecated_no_top_level_conftest(
+def test_pytest_plugins_in_non_top_level_conftest_unsupported_no_top_level_conftest(
     testdir
 ):
     from _pytest.deprecated import PYTEST_PLUGINS_FROM_NON_TOP_LEVEL_CONFTEST
@@ -160,8 +158,6 @@ def test_pytest_plugins_in_non_top_level_conftest_deprecated_no_top_level_confte
     subdirectory.mkdir()
     testdir.makeconftest(
         """
-        import warnings
-        warnings.filterwarnings('always', category=DeprecationWarning)
         pytest_plugins=['capture']
     """
     )
@@ -175,16 +171,14 @@ def test_pytest_plugins_in_non_top_level_conftest_deprecated_no_top_level_confte
     )
 
     res = testdir.runpytest_subprocess()
-    assert res.ret == 0
+    assert res.ret == 2
     msg = str(PYTEST_PLUGINS_FROM_NON_TOP_LEVEL_CONFTEST).splitlines()[0]
     res.stdout.fnmatch_lines(
-        "*subdirectory{sep}conftest.py:0: RemovedInPytest4Warning: {msg}*".format(
-            sep=os.sep, msg=msg
-        )
+        ["*{msg}*".format(msg=msg), "*subdirectory{sep}conftest.py*".format(sep=os.sep)]
     )
 
 
-def test_pytest_plugins_in_non_top_level_conftest_deprecated_no_false_positives(
+def test_pytest_plugins_in_non_top_level_conftest_unsupported_no_false_positives(
     testdir
 ):
     from _pytest.deprecated import PYTEST_PLUGINS_FROM_NON_TOP_LEVEL_CONFTEST