Micronucleus formation initiated by benzo[a]pyrene (B[a]P) and related xenobiotics is widely believed to reflect potential carcinogenic initiation, yet neither a dependence upon bioactivation nor the critical enzymes have been demonstrated. Using rat skin fibroblasts, protein oxidation (carbonyl formation) and content of prostaglandin H synthase (PHS) and cytochrome P4501A1 (CYP1A1) protein were determined by Western blot/immunodetection with enhanced chemiluminescence. DNA oxidation as 8-hydroxy-2'-deoxyguanosine formation was quantified using high-performance liquid chromatography with electrochemical detection. Fibroblast CYP1A1 activity assessed as ethoxyresorufin-O-deethylase was not detectable, and even CYP1A1 protein was measurable only after induction with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, TCDD additionally induced prostaglandin H synthase (PHS), which also was detectable constitutively. B[a]P 10 microM initiated the oxidation of DNA and protein, and the formation of micronuclei, all of which were enhanced over 2-fold by the dual CYP1A1/PHS inducer TCDD 10 nM, as well as by other PHS inducers, 12-O-tetradecanoylphorbol-13-acetate 1 microM and interleukin-1alpha 0.625 or 1.25 ng/ml, that do not induce CYP1A1 (p < .05). Conversely, B[a]P target oxidation and micronucleus formation were abolished by 1-aminobenzotriazole 1 mM (p < .05), which was a potent inhibitor of both peroxidases and P450. These results provide the first direct evidence that B[a]P-initiated micronucleus formation, like carcinogenic initiation, requires enzymatic bioactivation, and that peroxidase-dependent, reactive oxygen species-mediated oxidation of DNA, and possibly protein, constitutes a molecular mechanism of initiation in uninduced cells. Induction of either CYP1A1 or peroxidases such as PHS substantially enhances this genotoxic initiation, which may reflect cancer risk.