Gs alpha, the GTP-binding protein that mediates receptor stimulation of adenylyl cyclase, exists in small (Gs alpha-S) and large (Gs alpha-L) molecular forms. Previous work has established that these Gs alpha isoforms are products of alternatively spliced Gs alpha pre-mRNA. The present work examined the splicing pattern of Gs alpha mRNA in rat and human brain using a method based upon the polymerase chain reaction. In rat brain, transcripts encoding Gs alpha-L were most abundant and constituted 92% of total Gs alpha mRNA in hippocampus and cerebral cortex and 84% in striatum. Compared to rat brain, the relative abundance of Gs alpha-L mRNA was less in human brain and constituted 84% of total Gs alpha mRNA in hippocampus and cortex and 66% in striatum. In addition, it was found that approximately 40% of the Gs alpha-S mRNA utilizes an alternative splice site that encodes serine at amino acid 72. No regional variations in this splicing pattern were found.