Lipids of rat liver microsomes underwent peroxidation with production of malondialdehyde in the presence of H2O2 and hematin. Rates of peroxidation of 27-33 nmol of MDA formed/mg of microsomal protein/30 min were measured with 5 mM H2O2 and 10 microM hematin at 22 degrees C. Histidine (0.01 M) caused a 55% inhibition. Hematin could be added to the reaction mixtures either simultaneously with H2O2 or afterwards, when all H2O2 had been destroyed by catalase present in the microsomal preparation. Catalase was necessary for formation of MDA. Indeed, when heat-denatured microsomes were employed, incubation with H2O2 and the iron complex led to formation of lipid hydroperoxides; however, no production of MDA was observed, unless exogenous catalase was added together with H2O2 and hematin to the reaction mixture. The role of H2O2 in microsomal lipid peroxidation is that of promoting the formation of fatty acid hydroperoxides. These are decomposed in the presence of hematin, with formation of free radicals, bicyclic endoperoxides and MDA. Catalase is necessary to remove H2O2, which, after starting the peroxidation process, blocks the decomposition of lipid hydroperoxides, apparently by binding to the iron complex.