Diversion of bile pancreatic juice from the duodenum in rats stimulates cholecystokinin (CCK) release and pancreatic enzyme secretion. Intraduodenal perfusion of trypsin inhibits the release of CCK and pancreatic enzyme secretion. We hypothesized that the increased pancreatic enzyme secretion after pancreatic juice diversion is mediated by a trypsin-sensitive peptide secreted by the small intestine that stimulates release of CCK. To test this hypothesis, rats were surgically prepared with bile-pancreatic cannula and intestinal fistulas. Diversion of bile-pancreatic juice stimulated amylase output fivefold above basal and increased plasma CCK from a basal of 0.5 +/- 0.05 pM to 14 +/- 5 pM. Rapid perfusion (3 ml/min) of the duodenum with phosphate-buffered saline reversed the increase in amylase output and lowered the plasma CCK to 1.2 +/- 0.2. Administration of intestinal perfusate (3 ml/min) collected from a donor rat into the duodenum of a recipient rat with diversion of bile pancreatic juice increased amylase output threefold above basal and increased plasma CCK. The stimulatory activity of the intestinal perfusate was inactivated by treatment with trypsin but not by amylase or lipase. In addition, boiling did not alter the stimulatory activity of the intestinal perfusate. Perfusion of intestinal perfusate from donor rats pretreated with atropine did not stimulate amylase output and CCK release in recipient rats. By use of molecular membrane exclusion filters, stimulatory activity was retained (between 1,000 and 5,000). These results indicate that feedback regulation of pancreatic enzyme secretion is mediated by a CCK releasing peptide whose secretion from the duodenum is cholinergically mediated. This peptide is trypsin sensitive and has a molecular weight between 1,000 and 5,000.