The present study defines the origin of cholesterol subserving bile acid synthesis in male rats with an extracorporal bile duct by labeling newly formed cholesterol with tritiated water. Within 6 hr after interruption of the enterohepatic circulation, the bile acid pool was depleted. At this early time point the proportion from de novo cholesterol was 8% and 12% for biliary cholesterol and cholate, but 18% and 19% for muricholate and chenodeoxycholate, respectively. This proportion gradually rose to 40%, 34%, 51% and 51%, respectively, at 15 to 30 hr. At 78 hr after bile diversion, 64% of cholate was labeled, compared with 84% to 88% of the other biliary lipids and 71% of plasma cholesterol. Total and labeled bile acid secretion exhibited the same diurnal rhythm. To allow differentiation between direct hepatocytic de novo synthesis of bile acids from acetate and recycling of labeled plasma cholesterol, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (pravastatin) was infused from 54 to 78 hr. It suppressed total synthesis of primary bile acids by 60% to 80% but decreased the tritium label of bile acids only from a range of 74% to 92% (54 hr) to a range of 54% to 63% (78 hr), which was in the range of plasma cholesterol (58%). We conclude that bile acids and biliary cholesterol are synthesized mostly from preformed (i.e., plasma) cholesterol, both immediately after depletion of the pool in enterohepatic circulation and after derepression. Moreover, the hepatic cholesterol pools subserving the synthesis of different bile acids and biliary cholesterol secretion are not identical.