Data on the effect of vitamin E and selenium deficiency on lipid peroxidation in vivo have been limited. F2-isoprostanes are novel prostanoids that, free in plasma and esterified to phospholipids in tissues, are markers of lipid peroxidation in vivo. To address the importance of vitamin E and selenium in defense against lipid peroxidation in vivo, we determined F2-isoprostane concentrations in the plasma and organs of rats fed diets deficient in one or both nutrients. Weanling rats were fed a vitamin E- and selenium-deficient diet for 12 wk and then divided into four groups. One group continued to receive the doubly deficient diet, and the other three groups were fed the diet supplemented with vitamin E, selenium or both nutrients (control diet) for 4 wk. Plasma F2-isoprostanes in rats fed the doubly deficient diet were 5.2-fold higher than in animals changed to a control diet. In addition, there were significant differences in liver, lung, kidney, heart and skeletal muscle phospholipid-esterified F2-isoprostanes between these two groups. Lesser increases were noted in the group fed the vitamin E-deficient diet. Selenium deficiency alone was not associated with greater lipid peroxidation. Lipid peroxidation occurs in tissues of rats fed a vitamin E-deficient diet and is increased by concomitant selenium deficiency.