G subunits modulate several distinct molecular events involved with G protein signaling. In addition to regulating several effector proteins, G subunits help anchor G subunits to the plasma membrane, promote interaction of G with receptors, stabilize the binding of GDP to G to suppress spurious activation and provide membrane contact points for G protein-coupled receptor kinases. G subunits have also been shown to inhibit the activities of GTPase-activating proteins (GAPs), both phospholipase C-’s and RGS proteins, when assayed in solution under single-turnover conditions. We show here that G subunits inhibit G protein GAP activity during receptor-stimulated, steady-state GTPase turnover. GDP/GTP exchange catalyzed by receptor requires G in amounts approximately equimolar to G, but GAP inhibition was observed with super-stoichiometric G Potency of inhibition varied with the GAP and the G subunit, but half-maximal inhibition of the GAP activity of PLC-1 was observed with 5-10 nM G, which is at or below concentrations of G needed for regulation of physiologically relevant effector proteins. The kinetics of GAP inhibition of both receptor-stimulated GTPase activity and single-turnover, solution-based GAP assays suggested a competitive mechanism in which G competes with GAPs for binding to the activated, GTP-bound G subunit. An N-terminal truncation mutant of PLC-1 that cannot be directly regulated by G remained sensitive to inhibition of its GAP activity, suggesting that the G binding site relevant for GAP inhibition is on the G subunit rather than on the GAP. Using fluorescence resonance energy transfer between CFP- or YFP-labeled G protein subunits and Alexa532-labeled RGS4, we found that G directly competes with RGS4 for high-affinity binding to G_i-GDP-AlF₄.