The objectives of this work were to evaluate the effects of catechin on cytochrome P450 2E1 (CYP2E1)-dependent oxidative stress. Microsomes co-expressing human CYP2E1 with NADPH cytochrome P450 reductase and cytochrome b5 were incubated with NADPH and DTPA at pH 7.0. Superoxide anion generation was specifically detected by spin-trapping with DEPMPO. Generation of the DEPMPO-OOH adduct was not observed in the absence of CYP2E1 and in the presence of superoxide dismutase (SOD) or catechin, while catalase was ineffective. Reactive oxygen species generation was detected with 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) by the EPR-detection of its oxidation product, 3-carboxy-proxyl radical (CP). CP generation was not observed in the absence of CYP2E1 and in the presence of SOD, while catalase was ineffective. In contrast, catechin increased CPH oxidation, an effect that was not observed in the absence of CYP2E1 or in the presence of SOD (but not catalase), and was not associated with an increase in oxygen consumption. Catechin also increased the non-specific oxidation of the probes CPH and hydroethidine by the superoxide anion-generating system xanthine plus xanthine oxidase. Catechin oxidized CPH in the presence of horseradish peroxidase plus hydrogen peroxide, a catechin radical-generating system. In conclusion, catechin exhibits both antioxidant (superoxide-scavenging) and pro-oxidant effects under CYP2E1-dependent oxidative stress.