Most cells contain two forms of the alpha subunit of the G protein (Gs) that stimulates adenylate cyclase; their apparent molecular weights are 45,000 and 52,000. Two cDNAs that correspond to distinct mRNAs for the alpha subunit of Gs have been cloned from a bovine adrenal library and sequenced. The sequences of the two cDNAs, designated pGs-l and pGs-S, are identical except for a single stretch of 46 nucleotides in the coding region, where four are altered and 42 are deleted in pGs-S. Expression of pGs-S and pGs-l in COS-m6 cells yields protein products with apparent molecular weights of 45,000 and 52,000, respectively, based on their mobility in sodium dodecyl sulfate-polyacrylamide gels. We conclude that pGs-S and pGs-l encode the 45- and 52-kDa forms of Gs alpha, respectively, and propose that the mRNAs encoding these proteins arise from a single gene by internal alternative RNA splicing.