In dogs prepared with either pancreatic duct ligation (PDL) or chronic pancreatic fistula (PF), we investigated the effect of pancreatic enzymes on release of cholecystokinin (CCK) in response to corn oil in the upper small intestine. In control dogs without PDL, intraduodenal administration of corn oil (Lipomul containing 15 mmol of triglyceride) resulted in a marked and sharp increase in plasma CCK concentration during the 1st h, which then decreased during the following 1.5 h. In PDL dogs, on the other hand, no immediate increase occurred during the 1st 45 min after which a modest but gradual increase occurred in plasma CCK concentration during the following 2 h. Likewise, plasma CCK concentration increased significantly when Lipomul predigested with pancreatic enzymes or oleic acid was administered to PDL dogs. The increase was comparable to the response to Lipomul alone in control dogs. The infusion of Lipomul resulted in similar responses of plasma CCK in PF dogs as were found in PDL dogs when pancreatic juice was diverted to the exterior from the duodenum. The increases in plasma CCK paralleled the pancreatic protein secretion, and a significant correlation was found between plasma CCK concentration and the protein output. The present observations indicate that release of endogenous CCK by fat from the upper small intestine depends on an adequate digestion of neutral fat by pancreatic enzymes. Thus pancreatic enzymes play an important role on release of CCK in response to a neutral fat.