Pancreastatin (PST), a 49-amino acid polypeptide, inhibits endocrine and exocrine pancreatic functions. In this study, we examined the mechanism of the inhibitory action of PST on exocrine pancreatic secretion in the rat. In anesthetized rats prepared with pancreatic fistulas, intravenous administration of PST (500 pM.kg-1.h-1) completely inhibited 2-deoxyglucose (75 mg/kg)-stimulated amylase output to below basal levels. Because 2-deoxyglucose acts to stimulate the vagus, we assessed the ability of PST to inhibit carbachol-stimulated amylase release from isolated rat pancreatic acini. PST suppressed neither carbachol- nor cholecystokinin-stimulated amylase release, indicating that PST inhibits exocrine secretion via indirect mechanisms. To examine neural pathways for inhibition, we used pancreatic lobules to examine the action of PST on intrapancreatic neurons. Incubation of pancreatic lobules in 75 mM potassium buffer stimulated amylase release by a cholinergic pathway. PST dose dependently inhibited potassium-evoked amylase release, with maximal inhibition of 49.6 +/- 11%. In addition, when lobules were incubated with [3H]choline, PST inhibited KCl-stimulated release of synthesized [3H]acetylcholine by 43 +/- 5.7%. Other studies demonstrate that PST inhibits rat pancreatic enzyme secretion via presynaptic modulation of acetylcholine release.