Doc2 has two C2 domains that interact with Ca2+ and phospholipid. Munc13 has two C2 domains and one C1 domain that interacts with phorbol ester or diacylglycerol (DAG) and phospholipid. Both Doc2 and Munc13 are implicated in Ca2+-dependent neurotransmitter release, but their modes of action still remain unclear. We show here that Doc2 interacts with Munc13 both in a cell-free system and in intact PC12 cells during the high K+-induced Ca2+-dependent exocytosis. The Doc2-Munc13 interactions are stimulated by phorbol ester through the C1 domain of Munc13. Overexpression of the Doc2-interacting domain of Munc13 reduces the Ca2+-dependent exocytosis from PC12 cells, and co-expression with Doc2 suppresses this reduction. These results, together with the earlier findings that secretagogues produce DAG and elevate cytoplasmic Ca2+, suggest that the DAG-induced Doc2-Munc13 interactions play an important role in Ca2+-dependent exocytotic machinery.