Palmitoylation of the α 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 Gα 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 Gα z also inhibited its response to the GAP activity of Gα-interacting protein (GAIP), another RGS protein, and palmitoylation of Gα i1 inhibited its response to RGS4. The extent of inhibition of G z GAP, GAIP, RGS4, and RGS10 correlated roughly with their intrinsic GAP activities for the Gα 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.