Using a specific radioimmunoassay for cholecystokinin (CCK) we have studied the relation between circulating CCK concentrations and the feedback regulation of pancreatic enzyme secretion in conscious rats. Recirculation of diverted bile-pancreatic juice into the duodenum or intraduodenal perfusion of trypsin during biliary-pancreatic juice diversion produced basal output of amylase and trypsin and low portal CCK levels (less than 10 pmol/L). Biliary-pancreatic juice diversion or inactivation of trypsin caused increased CCK concentrations (peak values 50-100 pmol/L) and enzyme outputs. During biliary-pancreatic juice diversion, infusion of the CCK receptor antagonist proglumide suppressed the enzyme response without altering the increase in CCK. Measurement of portal and peripheral CCK during biliary-pancreatic juice diversion yielded values of 131 +/- 37 and 32 +/- 5 pmol/L, respectively. The peripheral CCK levels corresponded to concentrations achieved during exogenous CCK-8 infusion which resulted in similar enzyme outputs. Gel chromatography of portal plasma during diversion of biliary-pancreatic juice revealed one peak of CCK corresponding to CCK-8, and a larger peak eluted between CCK-33 and CCK-8, probably representing CCK-22. Similar CCK components were found in water extracts of jejunal mucosa, whereas the acetic acid extracts mainly contained CCK-33/39. We conclude that the negative feedback regulation of pancreatic enzyme secretion in rats is mediated by the release of CCK from the intestine and that the major molecular form of CCK in plasma is probably CCK-22.