A novel class of regulators of G protein signaling (RGS) proteins has been identified recently. Genetic evidence suggests that RGS proteins inhibit G protein–mediated signaling at the level of the receptor–G protein interaction or the G protein α subunit itself. We have found that two RGS family members, GAIP and RGS4, are GTPase-activating proteins (GAPs), accelerating the rate of GTP hydrolysis by G_iα1 at least 40-fold. All G_i subfamily members assayed were substrates for these GAPs; G_sα was not. RGS4 activates the GTPase activity of certain G_iα1 mutants (e.g., R178C), but not others (e.g., Q204L). The GAP activity of RGS proteins is consistent with their proposed role as negative regulators of G protein–mediated signaling.