Studying liver microsomes from 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced or vehicle-treated (noninduced) mice, we evaluated the in vitro effects of added chemicals on the production of reactive oxygen due to substrate/P450-mediated uncoupling. The catalase-inhibited NADPH-dependent H2O2 production (luminol assay) was lower in induced than noninduced microsomes. The effects of adding chemicals (2.5 μM) in vitro could be divided into three categories: Group 1, highly halogenated and coplanar compounds that increased H2O2 production at least 5-fold in induced, but not in noninduced, microsomes; Group 2, non-coplanar halogenated biphenyls that did not affect H2O2 production; Group 3, minimally halogenated biphenyls and benzo[a]pyrene that decreased H2O2 production. Molar consumption of NADPH and O2 and molar H2O2 production (o-dianisidine oxidation) revealed that Group 1 compounds mostly increased, Group 2 had no effect, and Group 3 decreased the H2O2/O2 and H2O2/NADPH ratios. Microsomal lipid peroxidation (thiobarbituric acid-reactive substances) was proportional to H2O2 production. Although TCDD induction decreased microsomal production of H2O2, addition of Group 1 compounds to TCDD-induced microsomes in vitro stimulated the second-electron reduction of cytochrome P450 and subsequent release of H2O2 production. This pathway is likely to contribute to the oxidative stress response and associated toxicity produced by many of these environmental chemicals.