Regulated secretion requires both calcium and MgATP. Studies in diverse secretory systems indicate that ATP is required to prime the exocytotic apparatus whereas Ca2+ triggers the final ATP-independent fusion event. In this paper, we examine the possible role of trimeric G proteins in these two steps of exocytosis in chromaffin cells. We show that in the presence of low concentrations of Mg2+, mastoparan selectively stimulates G proteins associated with purified chromaffin granule membranes. Under similar conditions in permeabilized chromaffin cells, mastoparan inhibits ATP-dependent secretion but is unable to trigger ATP-independent release. This inhibitory effect of mastoparan on secretion was specifically reversed by anti-Gαo antibodies and a synthetic peptide corresponding to the carboxyl terminus of Gαo. In contrast, mastoparan required millimolar Mg2+ for the activation of plasma membrane-bound G proteins and stimulation of ATP-independent secretion in permeabilized chromaffin cells. The latter effect was completely inhibited by anti-Gαi3 antibodies and a synthetic peptide corresponding to the carboxyl terminus of Gαi3. By confocal immunofluorescence and immunoreplica analysis, we provide evidence that in chromaffin cells Go is preferentially associated with secretory granules, while Gi3 is essentially present on the plasma membrane. Our findings suggest that these two trimeric G proteins act in series in the exocytotic pathway in chromaffin cells: a secretory granule-associated Go protein controls the ATP-dependent priming reaction, whereas a plasma membrane-bound Gi3 protein is involved in the late calcium-dependent fusion step, which does not require ATP.