Lipid peroxidation has been proposed to be a major mechanism involved in the pathophysiology of hepatic iron overload. Hepatic microsomal lipid peroxidation has been demonstrated in animals with dietary iron overload, and major products of lipid peroxidation with known cytotoxicity, such as malondialdehyde (MDA), may be involved in iron-induced hepatocellular injury by covalent binding to microsomal proteins. This investigation examined whether DBA/2Ibg mice fed a diet enriched with ferrocene-iron for 16 weeks, results in hepatic lipid peroxidation, and if liver microsomes contain proteins adducted by MDA. Chronic iron feeding to mice resulted in a severe hepatic iron overload with hepatic stores of iron 12-fold greater than those measured in control mice and a three-fold increase in hepatic concentrations of MDA, indicating the occurrence of iron-induced lipid peroxidation in vivo. Hepatic collagen content was increased by over three-fold (p < 0.05) in iron-fed mice as compared to control animals, suggesting increased fibrogenesis. Using rabbit antiserum specific for MDA amine protein adducts and immunoprecipitation-Western blotting, we documented formation of 10 liver microsomal proteins adducted by MDA in iron overload mice (approximate molecular weights; 214, 140, 129, 121, 103, 83, 62, 60, 48, and 43-kD). Control mice did not exhibit positive immunostaining for these protein adducts. The incubation of synthetic MDA with liver microsomes isolated from untreated mice demonstrated formation of MDA-adducted proteins with molecular weights comparable to those detected following in vivo iron overload. The data from this animal study are the first to demonstrate that lipid-derived aldehydes produced from hepatic iron overload in vivo, covalently bind and hence, chemically modify numerous proteins in microsomes. These data suggest that MDA modified proteins in microsomes may play a role in a sequence of events that lead to cell injury during metal-induced liver damage.