The molecular mechanisms underlying the Ca(2+) regulation of hormone and neurotransmitter release are largely unknown. Using a reconstituted [(3)H]norepinephrine release assay in permeabilized PC12 cells, we found that essential proteins that support the triggering stage of Ca(2+)-stimulated exocytosis are enriched in an EGTA extract of brain membranes. Fractionation of this extract allowed purification of two factors that stimulate secretion in the absence of any other cytosolic proteins. These are calmodulin and protein kinase Calpha (PKCalpha). Their effects on secretion were confirmed using commercial and recombinant proteins. Calmodulin enhances secretion in the absence of ATP, whereas PKC requires ATP to increase secretion, suggesting that phosphorylation is involved in PKC- but not calmodulin-mediated stimulation. Both proteins modulate release events that occur in the triggering stage of exocytosis. The half-maximal increase was elicited by 3 nM PKC and 75 nM calmodulin. These results suggest that calmodulin and PKC increase Ca(2+)-activated exocytosis by directly modulating the membrane- or cytoskeleton-attached exocytic machinery downstream of Ca(2+) elevation.