Rats were fed Chow (C; low-fat control) or a purified high-fat (30% of calories) low cholesterol diet containing menhaden oil (MO), corn oil (CO) or lard (L) for 2, 4 or 6 weeks. Rats were killed after an overnight fast. MO-fed rats had a larger weight/body weight that was accompanied by a lower mg liver DNA/g liver but unchanged liver DNA/body weight, indicating that hepatomegaly in the MO-fed rats was due to cellular hypertrophy. MO-feeding prevented the rise in plasma triacylglycerol and cholesterol observed with the other high-fat diets. There was a marked progressive accumulation of total liver triacylglycerol in the MO- and CO-fed rats. Plasma insulin was reduced in the MO-fed rats relative to all other groups. There were strong positive relationships between plasma insulin and triacylglycerol and between insulin and cholesterol in the high-fat-fed rats. Total liver glucose-6-phosphate dehydrogenase and malic enzyme activities were reduced by MO-feeding and were directly correlated with plasma cholesterol and insulin. These data are consistent with an apparent inhibition of hepatic triacylglycerol secretion by high-fat fish oil-feeding that is independent of the inhibitory effects on triacylglycerol synthesis. These data suggest a role for insulin in regulating the plasma triacylglycerol and cholesterol concentrations in MO-fed rats.