OBJECTIVE Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates protein and/or amylase secretion from isolated rat pancreatic acini. The effect of PACAP on pancreatic secretion in vivo and its mechanism of action were studied. METHODS Rats were prepared with pancreatic duct cannulation, pyloric ligation, and bile diversion into duodenum, and 2.5, 5, and 10 nmol/kg PACAP-27 was administered intravenously while pancreatic juice was collected for 30 minutes. In other groups of rats, the effect of 10 nmol/kg PACAP-27 was studied under the influence of either atropine; loxiglumide, an antisecretin serum; a combination of both loxiglumide and the antiserum; or a PACAP antagonist (PACAP 6-38). Plasma secretin and cholecystokinin concentrations were measured by radioimmunoassay. RESULTS (1) PACAP dose-dependently increased pancreatic secretion of fluid, bicarbonate, and protein; (2) the increase in pancreatic secretion paralleled that of plasma secretin and cholecystokinin; (3) a combination of loxiglumide and antisecretin serum eliminated the PACAP-stimulated pancreatic secretion, whereas loxiglumide or antisecretin serum alone partially but significantly blocked pancreatic secretion; (4) atropine failed to influence PACAP-induced pancreatic secretion; and (5) PACAP antagonist profoundly suppressed the PACAP action. CONCLUSIONS PACAP-27 dose-dependently stimulates pancreatic secretion of fluid, bicarbonate, and protein in rats. This effect is mediated by release of both secretin and cholecystokinin and is independent of cholinergic tone.