McCune-Albright syndrome (MAS) is a human genetic disorder caused by a mutation that constitutively activates the G(s)α subunit by abolishing GTP hydrolysis. MAS patients suffer from a range of endocrinopathies as well as polyostotic fibrous dysplasia of bone. We previously identified an intragenic suppressor of the MAS mutation in a yeast system, which substituted two residues in the GTP-binding site of Gpa1: L318P and D319V to suppress the constitutive activity of an R297H mutation, corresponding to the human F222P, D223V, and R201H mutations respectively. To extend these studies, the human GNAS gene was subjected to site-directed mutagenesis. Constructs expressing the MAS mutation (R201H), the MAS mutation plus the mutations homologous to the yeast suppressors (R201H, F222P/D223V), or the yeast suppressor mutation alone (F222P/D223V) were transfected into HEK293 cells, and basal and receptor-stimulated cAMP levels were measured. Expression of R201H increased the basal cAMP levels and decreased the EC(50) for hormone-stimulated cAMP production. These effects were dependent on the amount of R201H protein expressed. R201H, F222P/D223V abolished the constitutive activity of the MAS mutation and caused responses to hormone that were not different from those measured in cells expressing WT G(s)α. Interestingly, F222P/D223V behaved similar to R201H in causing increases in basal cAMP production, thus demonstrating constitutive activity. Substitution of another acidic (E) or polar (N, T, and G) amino acid at position 223 caused no suppression of R201H activity, while substitution of a second nonpolar amino acid (A) at this position partially suppressed, and the larger polar I residue completely suppressed the effects of R201H.