The role of endogenously activated protein kinase C in calcium-activated exocytosis was examined in digitonin-permeabilised bovine adrenal chromaffin cells. Protein kinase C activity was reduced by down-regulation following long-term treatment with PMA or by using the inhibitor sphingosine. Both treatments resulted in a substantial reduction in catecholamine secretion elicited by micromolar calcium, indicating that endogenous activation of protein kinase C is a major requirement for calcium-activated exocytosis in chromaffin cells.