[3H]palmitate attached to mutationally activated alpha s (alpha s-R201C) turns over rapidly, compared with palmitate linked to normal alpha s (t1/2 approximately 2 min versus 90 min); although alpha s-R201C (unlike normal alpha s) is predominantly found in the cytosol, [3H]palmitate is linked only to the membrane-bound pool of alpha s, normal or mutant. Similarly, activation of wild-type alpha s by isoproterenol, a beta-adrenoceptor agonist that also induces membrane-to-cytosol translocation of alpha s, dramatically accelerates depalmitoylation of alpha s. Thus, activation-induced removal of palmitate provides an explanation for activation-induced shifts of alpha s to the cytosol. Regulated palmitoylation cycles provide a potential general mechanism for controlling reversible changes in the cellular location and activity of a protein.