H epatic cholesterol metabolism was studied in rats fed purified diets supplemented (9% wt/wt) with either fish oil (FO) (n-3 fatty acids) or corn oil (CO) (n-6 fatty acids) for 4 wk. Rats were equipped with permanent catheters in heart, bile duct, and duodenum to allow studies under normal feeding conditions. [3H]-cholesteryl oleate-labeled small unilamellar liposomes, which are rapidly endocytosed by hepatocytes, were intravenously injected to label intrahepatic cholesterol pools, and plasma and bile were collected. FO as compared to CO induced a lowering of plasma cholesterol levels by 38% and of triglyceride levels by 69%. This reduction in plasma lipids in FO rats was accompanied by: (a) an increased bile acid pool size (28%); (b) a fourfold increase in the ratio cholic acid/chenodeoxycholic acid in bile; (c) increased biliary excretion of cholesterol (51%); (d) accelerated excretion of endocytosed free cholesterol into bile; (e) accelerated incorporation of endocytosed cholesterol in bile acids; (f) a significant increase in the bile acid-independent fraction of bile flow; and (g) a threefold increase in hepatic alkaline phosphatase activity. The results show that FO induces changes in transport and metabolic pathways of cholesterol in the rat liver, which result in a more rapid disposition of plasma-derived cholesterol into the bile.