The alpha subunit of the heterotrimeric Gs protein that couples the beta-adrenergic receptor to adenylyl cyclase undergoes post-translational palmitoylation. We examined the dynamics of this modification of alpha s by metabolic labeling of COS and S49 lymphoma cells under different conditions. The endogenous alpha s proteins were immunoprecipitated with a peptide-specific antibody, separated by SDS-polyacrylamide gel electrophoresis, and analyzed by fluorography and densitometry. A pulse-chase study of COS cells incubated with [3H]palmitate or [35S]methionine showed that for alpha s the palmitate turnover (t1/2 approximately 50 min) was significantly faster than the protein degradation. Treatment of cells with 10 microM isoproterenol, a beta-adrenergic receptor agonist, in the presence of [3H]palmitate led to a rapid 4-10-fold increase in the palmitoylation of alpha s. This increase in palmitoylation was concentration-dependent (EC50 approximately 0.9 microM) and blocked by the antagonist propranolol. The mutant alpha s proteins in the unc and H21a S49 cell lines did not show an increase in [3H]palmitate incorporation with isoproterenol treatment. Cholera toxin treatment of COS cells increased the [3H]palmitate incorporation into the alpha s subunits. These data indicate that palmitoylation of the alpha s subunit is dynamic and regulated by activation of the alpha s subunit.