One or more of three Gi proteins, Gi1-3, mediates hormonal inhibition of adenylyl cyclase. Whether this inhibition is mediated by the alpha or by the beta gamma subunits of Gi proteins is unclear. Mutations inhibiting the intrinsic GTPase activity of another G protein, the stimulatory regulator of adenylyl cyclase (Gs), constitutively activate it by replacing either of two conserved amino acids in its alpha subunit (alpha s). These mutations create the gsp oncogene which is found in human pituitary and thyroid tumours. In a second group of human endocrine tumours, somatic mutations in the alpha subunit of Gi2 replace a residue cognate to one of those affected by gsp mutations. This implies that the mutations convert the alpha i2 gene into a dominantly acting oncogene, called gip2, and that the mutant alpha i2 subunits are constitutively active. We have therefore assessed cyclic AMP accumulation in cultured cells which stably or transiently express exogenous wild-type alpha i2 complementary DNA or either of two mutant alpha i2 cDNAs. The results show that putatively oncogenic mutations in alpha i2 constitutively activate the protein's ability to inhibit cAMP accumulation.