The crystal structure of G(S alpha), the heterotrimeric G protein alpha subunit that stimulates adenylyl cyclase, was determined at 2.5 Angstrom in a complex with guanosine 5’-O-(3-thiotriphosphate) (GTP gamma S). G(S alpha) is the prototypic member of a family of GTP-binding proteins that regulate the activities of effectors in a hormone-dependent manner. Comparison of the structure of G(S alpha).GTP gamma S with that of G(i alpha).GTP gamma S suggests that their effector specificity is primarily dictated by the shape of the binding surface formed by the switch II helix and the alpha 3-beta 5 loop, despite the high sequence homology of these elements. In contrast, sequence divergence explains the inability of regulators of G protein signaling to stimulate the GTPase activity of G(S alpha). The beta gamma binding surface of G(S alpha) is largely conserved in sequence ana structure to that of G(i alpha), whereas differences in the surface formed by the carboxyl-terminal helix and the alpha 4-beta 6 loop may mediate receptor specificity.