The inhibitory glycine receptor (GlyR) in mammalian spinal cord displays pharmacological and molecular heterogeneity of its strychnine binding alpha subunit. Here, cDNAs were isolated which encode a variant (alpha ins 1) of the rat GlyR alpha 1 subunit that contains eight additional amino acids in its putative cytoplasmic domain. Analysis of the corresponding genomic sequence showed that alpha ins 1 transcripts result from alternative splice acceptor site selection. S1 nuclease protection experiments, Northern blot analysis, and RNA amplification by polymerase chain reaction revealed alpha 1 and alpha ins 1 mRNA in postnatal spinal cord, but not in other brain regions. Expression of synthetic alpha ins 1 RNA in Xenopus oocytes generated glycine-gated strychnine-sensitive chloride channels. These data indicate that alternative splicing contributes to GlyR alpha subunit heterogeneity in the mammalian central nervous system.