In order to define the source of cholesterol for bile acid synthesis and biliary cholesterol, hamsters with an extracorporeal bile duct received an intraperitoneal bolus of [3H]water labeling newly synthesized cholesterol. Thereafter the enterohepatic circulation was interrupted and a nutrient solution was infused during the experimental period of 78 h. In a separate group, pravastatin was administered (54-78 h) to allow discrimination of 3H-labeled cholesterol recycling from plasma and newly synthesized hepatic cholesterol late during the experiment. In controls, newly synthesized biliary cholesterol and primary bile acids derived from cholesterol newly synthesized during the experiment amounted to 5% and 12% immediately after depletion of the bile acid pool (6-9 h), respectively. After longterm bile diversion these proportions increased to 56-63%, whereas 71% of plasma cholesterol was labeled. Pravastatin inhibited the secretion of biliary cholesterol, cholate, and chenodeoxycholate by 30, 50, and 44%, respectively. In contrast, the preinfusion tritium label was suppressed by a maximum of 16%, 14%, and 26%, respectively, reflecting the contribution of cholesterol newly synthesized in the hepatocyte as opposed to labeled cholesterol recycling from the plasma. It is concluded that in the hamster newly synthesized cholesterol is of minor importance as substrate for bile acid synthesis as well as biliary cholesterol, both under near physiologic conditions and after long-term bile diversion. Moreover, the hepatic cholesterol pools subserving the synthesis of the primary bile acids are identical but appear to be different from that of biliary cholesterol directly after the depletion of the enterohepatic bile acids.