Chromaffin cells of the adrenal medulla have an important role in the sympathetic stress response. They secrete catecholamines and other hormones into the bloodstream upon stimulation by the neurotransmitter pituitary adenylate cyclase-activating polypeptide (PACAP). PACAP causes a long-lasting and robust secretory response from chromaffin cells. However, the cellular mechanisms by which PACAP causes secretion remain unclear. Our previous work showed that the secretory response to PACAP relies on signaling through phospholipase C epsilon (PLCε). The objective of this study was to clarify the role of signaling events downstream of PLCε. Here, it is demonstrated that a brief exposure of chromaffin cells to PACAP caused diacylglycerol (DAG) production-a process that was dependent on PLCε activity. DAG then activated protein kinase C (PKC), prompting its redistribution to the plasma membrane. PKC activation was important for the increases in cytosolic Ca(2+) and exocytosis that were evoked by PACAP. Indeed, pharmacological inhibition of PKC with NPC 15437, a competitive inhibitor of DAG binding, significantly disrupted the secretory response. NPC 15437 application also eliminated PACAP-stimulated effects on the readily releasable pool size, the Ca(2+) sensitivity of granule fusion, and the voltage dependence of Ca(2+) channel activation. Quantitative PCR revealed PKCβ, PKCε, and PKCμ to be highly expressed in the mouse chromaffin cell. Genetic knockdown of PKCβ and PKCε disrupted PACAP-evoked secretion, while knockdown of PKCμ had no measurable effect. This study highlights important roles for PKC signaling in a highly regulated pathway for exocytosis that is stimulated by PACAP.