Excess iron (Fe) intake has been associated with an increased risk of cardiovascular disease in humans, presumably the result of increased oxidative stress. Previous work by us has shown that feeding a high-Fe diet to selenium (Se)-deficient weanling mice for 4 wk resulted in elevated plasma cholesterol and triglycerides and increased hepatic thiobarbituric acid reactive substances (TBARS). Here, we report the effect of Fe overload in mice lacking cellular glutathione peroxidase (GPX1 knockout [KO] mice), the selenoenzyme thought to account for much of the antioxidant action of Se. Four groups of 9-13 weanling wild-type (WT) or GPX1 KO mice were randomly assigned, then fed either an Fe-adequate (35 ppm Fe) or high-Fe (1100 ppm Fe) casein-based diet for 4 wk. Iron was added as ferric citrate. Both diets also contained 0.2 ppm Se added as sodium selenite. As expected, liver GPX1 activity was essentially absent in the KO mice. Another Se parameter measured (hepatic thioredoxin reductase activity) did not vary across groups. Although liver Fe was elevated in mice fed the high-Fe diet, liver TBARS was largely unaffected either by mouse genotype or diet fed. Moreover, plasma lipids were not elevated in the Fe-over-loaded GPX1 KO mice. Thus, decreased GPX1 activity cannot account for the pro-oxidant hyperlipidemic effects observed earlier in mice fed the high-Fe Se-deficient diet. This suggests that impairment of Se functions other than GPX1 activity may be responsible for the elevated plasma lipids and hepatic TBARS seen in the Fe-overloaded Se-deficient mice.