We hypothesized that somatostatin exerts its inhibitory action on cholecystokinin (CCK) release and pancreatic secretion by inhibiting the secretion and/or action of a CCK-releasing peptide (CCK-RP) secreted from the small intestine. Our studies demonstrated that intravenous infusion of somatostatin (25 micrograms.kg-1.h-1) completely inhibited the increase in amylase output evoked by diversion of bile-pancreatic juice and 80 +/- 10% of the increase in plasma CCK. Intraduodenal administration of concentrated intestinal perfusate containing the CCK-RP collected from a donor rat with bile-pancreatic juice diversion raised amylase output by 2.3-fold and elevated plasma CCK levels to 7 +/- 0.8 pM in a recipient rat. The stimulatory effect of the concentrated intestinal washings was abolished when the "donor" rat was pretreated with somatostatin. In addition, in somatostatin-treated "recipient" rats, intraduodenal administration of intestinal washings containing CCK-RP also failed to elicit an increase in plasma CCK and amylase secretion. Furthermore, using duodenal mucosal explants, we demonstrated that the inhibitory action of somatostatin on CCK release evoked by CCK-RP was antagonized by pretreatment with pertussis toxin. These observations strongly suggest that somatostatin inhibits feedback regulation of pancreatic enzyme secretion by inhibiting both the secretion and action of CCK-RP.