In mammals, cholecystokinin regulates pancreatic exocrine secretion under physiological conditions. Cholecystokinin is released into the blood by several nutrients, but pancreatic enzyme secretion is not stimulated by nutrients in avian species. Furthermore, cholecystokinin at physiological concentrations does not induce pancreatic amylase secretion in birds in vitro. Acetylcholine and vasoactive intestinal polypeptide produced a concentration-dependent increase in amylase secretion at physiological concentrations. The combination of acetylcholine and vasoactive intestinal polypeptide produced an additive response in amylase secretion. Sodium nitroprusside, a spontaneous nitric oxide releaser, and bombesin, induced amylase secretion at concentrations greater than 10 nM and 100 nM, respectively. Gastrin and secretin increased amylase secretion at pharmacological concentrations. In conclusion, neural regulation by acetylcholine and vasoaactive intestinal polypeptide is important for pancreatic enzyme secretion in birds and the contribution of gut hormones seems to be physiologically unimportant.