A cDNA clone encoding a stimulatory G-protein alpha subunit (Gs alpha) was isolated from a cDNA library derived from cultured rat astrocytes. The nucleotide sequence of the cDNA indicated that it corresponds to the Gs alpha-2 form of Gs alpha mRNA, one of four Gs alpha mRNAs known to be derived by alternative splicing from the human Gs alpha gene. A ribonuclease protection assay using cRNA from this clone allowed distinction between the Gs alpha-1 and Gs alpha-2 mRNAs, which encode the 52-kDa (Gs-L) forms of Gs alpha. Astrocytes express relatively high amounts of Gs alpha-1 mRNA, much lower amounts of the Gs alpha-2 mRNA, and no detectable amounts of the mRNAs (Gs alpha-3 and Gs alpha-4) encoding the two 45-kDa forms of Gs alpha (Gs alpha-S). Similar results were obtained with RNA samples isolated from whole brain. The 45-kDa form of Gs alpha protein was not detectable by immunoblot analysis of a membrane preparation from rat cerebral cortex (the source of the astrocyte cultures). These results indicate that the expression of Gs alpha forms in astrocytes is similar to that found in whole brain.