The trypsin level in bile was studied by radioimmunoassay in a prospective series of 63 patients with gallstone disease but without signs or symptoms of cholecystitis or pancreatitis in order to find indirect evidence of a retrograde flow of pancreatic juice. Mobile duct stones were present in 18 patients and impacted stones in 12. The remaining 33 patients had stones only in the gallbladder and served as controls. The average intraoperative trypsin level of the ductal bile was normal, both in the control group and in the group with stones occluding a potential retrograde reflux of pancreatic juice. After removal of the impacted stones, the bile showed a significantly higher trypsin level. The average intraoperative trypsin level for the group with mobile stones was significantly higher than that of the control group, and was further increased 10 days postoperatively. The trypsin level of ductal bile from 23 of the 30 patients (77%) with bile duct stones exceeded that of the 33 patients with stone-free bile ducts, indicating an inflow of pancreatic juice to the bile ducts of patients with bile duct stones. The present results correspond well to those in a previous report on retrograde phasic contractions of the sphincter of Oddi in the majority of patients with bile duct stones. This dysfunction of the sphincter, which persisted for 10 days after surgical stone removal, may be the primary disorder, probably consisting of a retrograde propulsive activity of the sphincter of Oddi.