GTP hydrolysis by guanine nucleotide-binding proteins, an essential step in many biological processes, is stimulated by GTPase-activating proteins (GAPs). The mechanisms whereby GAPs stimulate GTP hydrolysis are unknown. We have used mutational, biochemical, and structural data to investigate how RGS4, a GAP for heterotrimeric G protein α subunits, stimulates GTP hydrolysis. Many of the residues of RGS4 that interact with Giα1are important for GAP activity. Furthermore, optimal GAP activity appears to require the additive effects of interactions along the RGS4-Gαinterface. GAP-defective RGS4 mutants invariably were defective in binding Gαsubunits in their transition state; furthermore, the apparent strengths of GAP and binding defects were correlated. Thus, none of these residues of RGS4, including asparagine 128, the only residue positioned at the active site of Giα1, is required exclusively for catalyzing GTP hydrolysis. These results and structural data (Tesmer, J. G. G., Berman, D. M., Gilman, A. G., and Sprang, S. R. (1997) Cell89, 251–261) indicate that RGS4 stimulates GTP hydrolysis primarily by stabilizing the transition state conformation of the switch regions of the G protein, favoring the transition state of the reactants. Therefore, although monomeric and heterotrimeric G proteins are related, their GAPs have evolved distinct mechanisms of action.