The antitumor drug, adriamycin, enhances NADPH-dependent lipid peroxidation in liver microsomes via the formation of superoxide anion radicals (O2) and hydrogen peroxide (H2O2). In the presence of metal ions additional reactive species are generated, causing stimulation of lipid peroxidation. However, in this study it was found that the stimulation of NADPH-dependent lipid peroxidation by adriamycin was not only affected by the production of O2 and H2O2. Adriamycin also enhances the catalysis by metal ions of the formation of those reactive oxygen species which initiate peroxidation. This was inferred from the fact that adriamycin stimulated malondialdehyde production at low ferrous ion concentrations, whereas at high ferrous ion concentrations no stimulation was found. Additional evidence was found in experiments in which the enzymic redox cycle of adriamycin in microsomes was abolished by heat-inactivation of the microsomes, and O2 and H2O2 were only produced with xanthine and xanthine oxidase. In this case in the presence of ferrous ions, adriamycin stimulated lipid peroxidation.