The hypothesis that the absence of pancreatic secretions from the lumen of the intestinal tract could alter the secretion rate of biliary lipids and the kinetics of bile salts was tested in rats with a pancreatic fistula fed intragastrically either a high or a low fat liquid diet over a 4-day period. In animals with external derivation of all pancreatic secretions who were given the high fat diet, stools were pale and bulky, the basal synthesis rate of bile acids was almost twice that of controls, and there was an increase in the secretion rate of cholesterol and phospholipids calculated from the first 2 h of bile collection. On the low fat diet, there was no change in basal bile salt synthesis, but the pool was larger and the secretion rate of bile acids and phospholipids was increased. A study of the quantitative relationship between the excretion rate of phospholipids and of cholesterol to that of bile acids following complete biliary diversion revealed a disproportionately large output of cholesterol at low bile acid flow rates under both diets in rats with a pancreatic fistula. These data suggest that experimental pancreatic insufficiency leads to alterations of the secretion rate of biliary lipids and to uncoupling of the close relationship between bile acids and cholesterol.