We examined the effect of cholecystokinin (CCK) on the receptors for vasoactive intestinal peptide (VIP) and secretin in rat pancreatic acini. CCK decreased the specific binding of 125I-VIP and 125I-secretin by 42 and 51%, respectively. This CCK-induced inhibition was caused by an apparent decrease in the capacity of high-affinity binding sites of VIP and secretin receptors. CR 1409, a specific antagonist of CCK, abolished CCK-induced binding inhibition, whereas 12-O-tetradecanoylphorbol-13-acetate, A23187, and cycloheximide did not affect the binding of the radioligands. Both N2,O2-dibutyryl guanosine 3',5'-cyclic monophosphate (Bt2cGMP) and nitroprusside inhibited the specific binding of 125I-VIP. This inhibition, however, was because of an apparent decrease in the capacity of low-affinity binding sites on VIP receptors. CCK-induced downregulation of VIP and secretin receptors was associated with the diminished acinar response to VIP or secretin-induced adenosine 3',5'-cyclic monophosphate accumulation and amylase secretion, whereas neither Bt2cGMP nor nitroprusside affected VIP-induced amylase secretion. Data suggest that CCK-induced downregulation is mediated by the initial interaction of CCK with CCK receptors followed by some postreceptor process, which appears unrelated to protein kinase C, calcium mobilization, decrease in protein synthesis, or cellular cGMP increases. This downregulation, at least in part, accounts for CCK-induced restricted stimulation of amylase secretion by VIP and secretin.