XLalpha(s), the large variant of the stimulatory G protein alpha subunit (Gsalpha), is derived from GNAS1 through the use of an alternative first exon and promoter. Gs(alpha) and XLalpha(s) have distinct amino-terminal domains, but are identical over the carboxyl-terminal portion encoded by exons 2-13. XLalpha(s) can mimic some functions of Gs(alpha), including betagamma interaction and adenylyl cyclase stimulation. However, previous attempts to demonstrate coupling of XLalpha(s) to typically Gs-coupled receptors have not been successful. We now report the generation of murine cell lines that carry homozygous disruption of Gnas exon 2, and are therefore null for endogenous XLalpha(s) and Gs(alpha) (Gnas(E2-/E2-)). Gnas(E2-/E2-) cells transfected with plasmids encoding XLalpha(s) and different heptahelical receptors, including the beta2-adrenergic receptor and receptors for PTH, TSH, and CRF, showed agonist-mediated cAMP accumulation that was indistinguishable from that observed with cells transiently coexpressing Gs(alpha) and these receptors. Our findings thus indicate that XLalpha(s) is capable of functionally coupling to receptors that normally act via Gs(alpha).