The stimulatory GTP-binding protein (Gs) of the uncoupled mutant of S49 lymphoma cells is deficient in its ability to transduce hormonal signals from ligand-bound beta-adrenergic receptors to the catalytic component of adenylate cyclase. In order to define the genetic defect in the Gs of uncoupled S49 cells, a complementary DNA clone encoding the alpha-subunit of Gs was analyzed and the deduced primary structure of the defective subunit compared to that of the wild-type subunit. A single nucleotide transversion was found that coded for a proline rather than an arginine at residue 389. The results indicate a domain of the alpha-subunit of Gs that specifically interacts with hormone receptors.