The peripheral membrane protein gephyrin co-purifies with the inhibitory postsynaptic glycine receptor (GlyR) of mammalian spinal cord. By immunoelectron microscopy, gephyrin has been localized at the cytoplasmic face of glycinergic postsynaptic membrane specializations. Here, we used specific monoclonal antibodies to demonstrate the presence of gephyrin in all regions of rat brain known to contain synapses and compared its histochemical distribution with that of GlyR antigens. In most brain structures, gephyrin is expressed independently of the GlyR alpha 1 subunit, but its distribution is very similar to the pattern obtained with mAb 4a, a monoclonal antibody recognizing the known GlyR alpha and beta subunits. Our data suggest a much wider distribution of gephyrin and GlyR proteins in the mammalian CNS than anticipated previously.