A bioassay for studying the cholecystokinin (CCK)-releasing activity of intraluminal protease-sensitive bioactive peptides was developed. In conscious rats, bile and pancreatic juice were chronically diverted from the proximal intestine to the ileum to cause chronic stimulation of CCK release and pancreatic protein secretion. CCK-releasing activity of test substances was assayed during transient inhibition of CCK release by intraduodenal sodium taurocholate (78 mumols/h). Intestinal secretion as a source of the putative trypsin-sensitive intestinal CCK-releasing peptide was obtained by rapid intestinal perfusion of isolated Thiry-Vella fistulae of jejunum in conscious rats, collected with or without atropine pretreatment. Partially purified rat pancreatic secretory trypsin inhibitor (PSTI, or "monitor peptide") was compared with ovomucoid trypsin inhibitor (OMTI) and with concentrated jejunal secretions for CCK-releasing activity and trypsin inhibitor activity. Concentrated, heat-treated jejunal secretions were the strongest stimulants of CCK release and pancreatic protein secretion in this model. OMTI had no CCK-releasing activity in this model, whereas a larger amount (approximately 5x, based on trypsin inhibitor activity) of PSTI weakly but significantly stimulated CCK release. CCK-releasing activity manifested by pancreatic protein secretion was equivalent in intestinal washes from atropine-treated and control Thiry-Vella fistula donor rats. Concentrated jejunal secretions had no trypsin inhibitory activity, indicating that the putative intestinal CCK-releasing peptide and "monitor peptide" are different substances.