The signaling mechanisms underlying the insulinotropic effect of the pancreatic neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) were studied in insulin-producing HIT-T15 cells. It was found that unlabeled PACAP38 dose-dependently displaced [125I]PACAP27 from HIT-T15 cell membranes, showing binding of the peptide to these cells. Furthermore, at levels above 0.1 nM, PACAP38 stimulated insulin secretion in a Ca2+ -dependent manner, requiring 0.5 mM glucose. The peptide also markedly increased the cellular cAMP content and slightly stimulated the formation of inositol phosphates. Moreover, PACAP38 elevated the cytoplasmic Ca2+ concentration ([Ca2+]cyt) in fura-2-AM-loaded cell suspensions. The PACAP38-induced increase in cellular cAMP content was also seen at zero glucose, whereas the increases in insulin secretion and [Ca2+]cyt were abolished by removal of glucose or extracellular Ca2+. We conclude that PACAP38 binds to HIT-T15 cell membranes and acts in a glucose-dependent manner on adenylate cyclase to form cAMP, which both directly and indirectly, through increased [Ca2+]cyt, stimulates exocytosis.