The purpose of this study was to determine the effects of dietary fat, vitamin E and iron on oxidative damage and antioxidant status. Male Swiss-Webster mice (1 mo old) were fed a basal vitamin E-deficient diet that contained either 8% fish oil + 2% corn oil or 10% lard with or without 1 g dl-alpha-tocopheryl acetate. The diets without vitamin E contained either 0.21 or 0.95 g ferric citrate/kg. Diets were fed for 4 wk/kg diet. Compared with the vitamin E-supplemented groups, mice fed diets without vitamin E (with or without supplemental iron) had significantly (P < 0.05) higher hepatic levels of thiobarbituric acid-reactive substances (TBARS), conjugated dienes and protein carbonyls when they were fed fish oil, but not lard. The levels of TBARS were further increased by iron supplementation in the mice fed fish oil. Significantly lower concentrations of alpha-tocopherol and higher glutathione (GSH) were found in the liver of mice fed fish oil and vitamin E than in those fed lard and vitamin E (P < 0.05). The activities of superoxide dismutase and glucose-6-phosphate dehydrogenase were lower in the fish oil-fed mice than in those fed lard (P < 0.05). The activities of Se-GSH peroxidase, non-Se-GSH peroxidase, catalase, and glutathione reductase were not altered by dietary fat or vitamin E/iron. The results obtained provide experimental evidence of the prooxidative effects of high dietary fish oil and iron, and suggest that vitamin E protects not only lipid-soluble compounds, but also water-soluble constituents, against oxidative damage. Further, dietary lipid plays a key role in determining cellular susceptibility to oxidative stress.