The functional consequences of the mutation of a conserved Cys-214 in Gα i1 have been investigated. We reported herein that substitutions of Cys-214 of Gα i1 to either alanine or tryptophan abolished the intrinsic GTPase activity. Free phosphate release from [ 32 P]GTP-bound Gα i1 C214A or [ 32 P]GTP-bound Gα i1 C214W was at least 30-fold lower than that of the wild-type Gα i1 in single-turnover GTPase assays. Consistently, tryptic proteolysis of C214A and C214W proteins showed that they were partially protected by GTP, further confirming that the GTPase activity in both mutant proteins was impaired. Expression of C214A or C214W mutants in Chinese hamster ovary K1 cells caused significant inhibition of forskolin-stimulated adenylate cyclase activity. However, the mutations did not significantly affect the GTP[S] (guanosine 5´-[γ-[ 35 S]thio]triphosphate)-binding activity. Both C214A and C214W mutants serve as good substrates for pertussis toxin-catalysed ADP ribosylation, indicating that they interact well with βγ subunits. Moreover, RGS4 protein, a GTPase-activating protein for Gα i1 , cannot interact with Cys-214 mutants even in the presence of AlF 4 − , which induces the transition state of Gα. In summary, our findings suggest that C214A or C214W are GTPase-deficient mutants and can functionally serve as constitutively active forms of Gα i1 in cells.