The heterotrimeric G proteins transduce extracellular signals by interacting with specific intracellular effectors. We have used a scanning mutagenesis approach to identify amino acids of alpha S, the alpha subunit of Gs, that determine the specificity of its interaction with its effector, adenylyl cyclase. In alpha subunit chimeras, residues 236-356 of alpha S comprise the shortest linear stretch that is required for activation of adenylyl cyclase. Within these 121 residues, we identified four clusters of residues in which substitutions prevented effector activation. Mutations in three of these regions did not affect alpha subunit expression or the GTP-induced conformational change. The identified alpha S residues in the NH2-terminal half of the 121-residue region endowed the cognate alpha i2 segment with the ability to activate effector, while those in the COOH-terminal half did not. In a three-dimensional G alpha model, based on the structure of p21ras, the effector-activating residues of alpha S form a surface on the membrane-facing side of the molecule; this surface includes a region that changes conformation upon binding GTP.