The effects of biliary diversion on pancreatic enzyme activities of intestinal contents was studied in conscious rats prepared with biliary and pancreatic fistulae. Diversion of bile from the intestine for 1 day caused on 80% decrease in trypsin and chymotrypsin activities of intestinal contents, in spite of increased (230%) pancreatic trypsin and chymotrypsin secretion. Bile diversion in fed rats caused a smaller decrease (58%) in trypsin and chymotrypsin activities of intestinal contents. Sodium taurocholate (100 mumol/hr intraduodenally) partially reversed the changes in pancreatic secretion and intestinal contents' activities of trypsin and chymotrypsin caused by bile diversion. The results indicated that bile was important in controlling the rate of disappearance of trypsin and chymotrypsin activities from the small intestine. The mechanism for this was studied by comparing the rate of disappearance of trypsin activity in vivo and in vitro. Bovine trypsin, with or without sodium taurocholate, was infused intraduodenally into conscious rats deprived of bile-pancreatic juice and the recovery of trypsin activity from the small intestine determined. Taurocholate increased recovery of trypsin from the small intestine more than threefold, but inactivation of bovine trypsin in vitro was not retarded by sodium taurocholate. The results indicate that bile in the small intestine controls the rate of disappearance of intraluminal trypsin and chymotrypsin activities, probably by inhibiting their autodigestion in vivo. We previously reported that bile duct ligation in rats caused decreased trypsin and chymotrypsin activities in the small intestine, but increased pancreatic enzyme secretion. We concluded that trypsin and chymotrypsin underwent accelerated inactivation in the small intestine in the absence of bile. The present study was designed to explore the mechanism for the effects of bile deprivation on intraluminal proteolytic enzyme activities in the rat.