Endocytosis represents an important mechanism regulating cell-surface expression of neurotransmitter receptors, including GABAA receptors, in neurons. Little is known, however, about trafficking of internalized receptors. Here, we used antibody tagging in living rat hippocampal neurons in culture to monitor GABAA receptor internalization. We show that cell-surface receptors have a homogeneous distribution reflecting their mobility in the membrane. Unexpectedly, internalized GABAA receptors were detected mainly in a subsynaptic pool associated with gephyrin at postsynaptic sites, whereas AMPA-type glutamate receptors were accumulated in the soma. This process was time-dependent and could be prevented by blocking clathrin-coated vesicle endocytosis. In control experiments, the existence of an intracellular pool of GABAA receptors associated with gephyrin was confirmed independently of internalization of surface receptors, and constitutive endocytosis, unrelated to antibody-tagging, could be demonstrated for both AMPA and GABAA receptors using a biotinylation assay. These results suggest that cycling of GABAA receptors between the cell surface and the subsynaptic pool provides a mechanism for the short-term regulation of GABAergic neurotransmission. Furthermore, the close association of gephyrin with internalized GABAA receptors suggests a role in intracellular receptor trafficking.