The liver microsomal ethanol-inducible cytochrome P-450 (P-450IIE1) form is known to exhibit a high rate of oxidase activity in the absence of substrate and it was therefore of interest to evaluate whether this form of P-450 could contribute to microsomal and liposomal NADPH-dependent oxidase activity and lipid peroxidation. The rate of microsomal NADPH-consumption, O₂⁻-formation, H₂O₂-production and generation of thiobarbituric acid (TBA) reactive substances correlated to the amount of P-450IIE1 in 28 microsomal samples from variously treated rats. Anti-P-450IIE1 IgG inhibited, compared to control IgG, microsomal H₂O₂-formation by 45% in microsomes from acetone- treated rats and by 22% in control microsomes. NADPH-dependent generation of TBA-reactive products was completely inhibited by these antibodies, whereas preimmune IgG was essentially without effect. Liposomes containing reductase and P-450IIE1 were peroxidized in a Superoxide dismutase (SOD) sensitive reaction at a 5–10-fold higher rate than membranes containing 3 other forms of cytochrome P-450. Lipid peroxidation in reconstituted vesicles dependent on the presence of P-450IIB1 was by contrast not inhibited by SOD. Microsomal peroxidase activities, using 15-(S)-hydroperoxy-5- cis-8,11,13-trans-eicosatetraenoic acid as a substrate were high in microsomes from phenobarbital- or ethanol-treated rats but low in membranes from isoniazid-treated rats, having the highest relative level of P-450IIE1. It is suggested that the oxidase activity of P-450IIE1 contributes to microsomal NADPH- dependent lipid peroxidation. The combined action of the oxidase activity by P-450IIE1 and the peroxidase activities by P-450IIB1 and other forms of P-450 may be important for the high rate of lipid peroxidation observed in e.g. microsomes from ethanol- or acetone-treated rats. The possible importance of cytochrome P-450IIE1-dependent lipid peroxidation in vivo after ethanol abuse is discussed.