The effect of highly purified ethyl all-cis-5,8,11,14,17-icosapentaenoate (EPA-E) on cholesterol metabolism in rats was examined to clarify the mechanism of its hypolipidemic action. Pretreatment with EPA-E reduced the increase in plasma radioactivity after oral administration of [14C]cholesterol. The conversion of [14C]3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to [14C]mevalonic acid was significantly inhibited in liver microsomes obtained from rats treated with EPA-E. There was an increase in free cholesterol and a marked rise in the eicosapentaenoic acid (EPA) content of phospholipids in these microsomes. EPA-E restored the suppression of biliary secretion induced by feeding a casein-rich diet to bile duct-cannulated rats. Furthermore, when serum lipoprotein (d < 1.210) from rats given EPA-E was i.v. injected into normal rats, a more rapid elimination of cholesterol was observed as compared to that in rats injected with lipoprotein from EPA-E-untreated rats. This rapid clearance was found in the lipoprotein fractions of d < 1.006 and 1.006 < d < 1.063. These findings suggest that EPA-E has an inhibitory effect on intestinal cholesterol absorption and hepatic cholesterol biosynthesis, and an enhancing effect on hepatic biliary secretion. EPA-E would also seem to cause modification of serum lipoproteins, whereby their clearance from the serum is increased.