The distribution of glycine receptor (GlyR)-associated gephyrin has been investigated in rat spinal cord neurons maintained in vitro by means of immunocytochemical techniques. Gephyrin, which is crucial for the stabilization of postsynaptic GlyR microdomains, is present in mature neurons at postsynaptic differentiations. With immunofluorescence, discontinuous patches of gephyrin were detected within the neuronal soma of spinal cord neurons on the 1st day after plating. Subsequently, gephyrin was present at membrane areas that correspond to points of contact between cells or with the culture dish. By the 5th day, gephrin was mostly associated with the MAP2-positive somatodendritic compartment. With immunoelectron microscopy, gephyrin blobs detected at the earliest stages (1-3 days after plating) were found within the cytoplasm or associated with the plasma membrane. Asymmetrically immunostained intercellular contacts were only detected after 5 days, and gephyrin was found in association with clearly differentiated postsynaptic membranes at 7 days. At later stages, we observed gephyrin immunoreactivity only at some synapses. Our results suggest that gephyrin accumulates initially at the locus of cell-to-cell contacts involved in adhesion processes. These localizations may define hot spots for later accumulation of the GlyR and possibly other receptors.