The peripheral membrane protein gephyrin copurifies with the inhibitory glycine receptor of mammalian spinal cord. It binds with high affinity to polymerized tubulin and has been implicated in the anchoring of the glycine receptor to cytoskeletal elements. Recently, cDNA cloning has identified variants of the gephyrin mRNA, which originate from alternative splicing of four exonic regions (cassettes 1-4). In this study, the expression patterns of gephyrin splice variants were determined in the adult and developing rat brain by in situ hybridization with synthetic oligonucleotide probes. Gephyrin transcripts were detected throughout the brain and spinal cord, with mRNAs containing cassette 2 (C2 transcripts) being predominant in adult animals. C3 and C4 transcripts were seen in cerebellar granule cells and in the dentate gyrus, whereas a C1 probe did not produce detectable hybridization signals. During development, C2 and C3 mRNAs were found in most brain regions. Generally, the spatial and temporal distribution of gephyrin transcripts is similar to that of the glycine receptor beta subunit mRNA reported previously.