The effect of atropine on the exocrine pancreatic secretory response to intestinally infused trypsin inhibitor and protein in conscious rats was investigated. Bile and pancreatic juice were collected and continuously returned to the intestine throughout all experiments. Ovomucoid trypsin inhibitor (OMTI) was infused at 1, 3, 6, 12, and 30 mg/h id, simultaneously with intravenously infused atropine (100 micrograms X kg-1 X h-1) or 0.15 M NaCl. Casein was infused at 300 mg/h id with or without intravenous atropine. Atropine inhibited basal pancreatic protein and fluid secretion 65.7 and 24.7%, respectively. Atropine had no effect on incremental (above basal) pancreatic protein and fluid output during infusion of maximally effective doses of OMTI (12 and 30 mg/h) and increased the incremental responses to submaximal doses of OMTI and to casein. The results are consistent with the hypothesis that cholecystokinin mediates negative feedback regulation by luminal proteases and that cholinergic mechanisms are not directly involved in this regulatory mechanism.