Palmitoylation of the alpha subunit of the guanine nucleotide-binding protein G(z) inhibited by more than 90 percent its response to the guanosine triphosphatase (GTPase)-accelerating activity of G(z) GAP, a G(z)-selective member of the regulators of G-protein signaling (RGS) protein family of GTPase-activating proteins (GAPs). Palmitoylation both decreased the affinity of G(z) GAP for the GTP-bound form of G alpha(z) by at least 90 percent and decreased the maximum rate of GTP hydrolysis. Inhibition was reversed by removal of the palmitoyl group by dithiothreitol. Palmitoylation of G alpha(z) also inhibited its response to the GAP activity of G alpha-interacting protein (GAIP), another RGS protein, and palmitoylation of G alpha(l1) inhibited ils response to RGS4. The extent of inhibition of G(z) GAP, GAIP, RGS4, and RGS10 correlated roughly with their intrinsic GAP activities for the G alpha target used in the assay. Reversible palmitoylation is thus a major determinant of G(z) deactivation after its stimulation by receptors, and may be a general mechanism for prolonging or potentiating G-protein signaling.