We have used stage-specific assays for MgATP-dependent priming and for Ca(2+)-activated triggering in the absence of free MgATP to examine the effects of alpha-SNAP, 14-3-3 proteins and calmodulin on regulated exocytosis in permeabilized adrenal chromaffin cells. All three proteins lead to a Ca(2+)-dependent increase in catecholamine secretion. Both alpha-SNAP and 14-3-3 proteins stimulated in a priming but not in a triggering assay. In contrast, calmodulin was stimulatory in triggering but not priming. The effects of alpha-SNAP and 14-3-3 proteins were likely to be due to distinct mechanisms of action since they differed in Ca(2+)-dependency, time course and extent of stimulation and their effects were additive. alpha-SNAP and 14-3-3 proteins did not appear to exert their priming action through changes in synthesis of phosphatidylinositol (4,5) bisphosphate. The data show that these three proteins have distinct stage-specific actions on exocytosis and indicate that alpha-SNAP acts in an early MgATP-requiring stage and not in the late Ca(2+)-triggered steps immediately prior to membrane fusion as previously suggested.