The effect of eicosapentaenoic acid (EPA) on fatty acid oxidation and on key enzymes of triglyceride metabolism and lipogenesis was investigated in the liver of rats. Repeated administration of EPA to normolipidemic rats resulted in a time-dependent decrease in plasma triglycerides, phospholipids and cholesterol. The triglyceride-lowering effect was observed after one day of feeding whereas lowering of plasma cholesterol and phospholipids was observed after five days of treatment. The triglyceride content of liver was reduced after two-day treatment. At that time, increased mitochondrial fatty acid oxidation occurred whereas mitochondrial and microsomal glycerophosphate acyltransferase was inhibited. The phosphatidate phosphohydrolase activity was unchanged. Adenosine triphosphate:citrate lyase, acetyl-CoA carboxylase, fatty acid synthetase and glucose-6-phosphate dehydrogenase were inhibited during the 15 d of EPA treatment whereas peroxisomal beta-oxidation was increased. At one day of feeding, however, when the hypotriglyceridemic effect was established, the lipogenic enzyme activities were reduced to the same extent in palmitic acid-treated animals as in EPA-treated rats. In cultured rat hepatocytes, the oxidation of [14C]palmitic acid to carbon dioxide and acid-soluble products was stimulated in the presence of EPA. These results suggest that the instant hypolipidemia in rats given EPA could be explained at least in part by a sudden increase in mitochondrial fatty acid oxidation, thereby reducing the availability of fatty acids for lipid synthesis in the liver for export, e.g., in the form of very low density lipoproteins, even before EPA induced peroxisomal fatty acid oxidation, reduced triglyceride biosynthesis and diminished lipogenesis.