Previous studies have shown that trypsin and chymotrypsin in the duodenum exert a negative-feedback regulation on pancreatic enzyme secretion in the rat. The mechanism responsible for this physiological phenomenon is unknown. By use of a specific and sensitive bioassay based on amylase release from isolated pancreatic acini, the role of cholecystokinin in the negative-feedback regulation of exocrine pancreatic secretion was examined. Rats were prepared with duodenal cannulas and pancreaticobiliary cannulas. Diversion of pancreaticobiliary juice resulted in a threefold increase in pancreatic protein output and an increase of plasma cholecystokinin from a basal level of 0.5 +/- 0.08 pM cholecystokinin octapeptide (CCK-8) to 16 +/- 4 pM CCK-8. Perfusion of trypsin (2 mg/h) or pancreaticobiliary juice returned pancreatic protein output to basal levels and plasma cholecystokinin to 2.1 +/- 1.2 and 0.33 +/- 0.1 pM, respectively. The inhibitory effect of trypsin on cholecystokinin release was enzyme and site specific, since inhibition of cholecystokinin release was not observed with perfusion of amylase into the duodenum or with trypsin into the ileum. Intravenous infusion of proglumide abolished the increase in pancreatic secretion following diversion of pancreaticobiliary juice. Intraduodenal perfusion of lidocaine, infusion of tetrodotoxin into the superior mesenteric artery, or intravenous infusion of atropine inhibited the rise in plasma cholecystokinin seen with diversion of pancreaticobiliary juice. These studies suggest that feedback regulation of pancreatic enzyme secretion in the rat is mediated by release of cholecystokinin. Furthermore, the feedback mechanism is neurally mediated, involving a cholinergic pathway.