Human cytochrome P450 1A1 (1A1) and microsomal epoxide hydrolase (mEH)-dependent metabolic activation of benzo[a]pyrene (BP) have been reconstituted with microsomes from yeast cells expressing the two enzymes. The formation of the postulated ultimate mutagen 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydro-BP, the so-called diol epoxide-2 (DE2) derived from the reoxidation of BP-7,8-dihydrodiol by 1A1, was estimated by HPLC measurement of its hydrolysis product 7 beta, 8 alpha, 9 alpha, 10 beta-tetrahydrotetrol-BP (T2-tetrol). The 1A1/mEH coupled system was analyzed by varying the incubation time, initial substrate concentration, and molar ratio of the two enzymes. A minimum kinetic model of BP metabolism by 1A1 and mEH was constructed on the basis of the overall kinetic parameters (Vmax, Km) for a number of individual steps determined with human 1A1 and mEH expressed in yeast. The model was converted into a set of differential equations including 30 independent kinetic constants, 15 chemical species, and 8 enzymes and enzyme/substrate complexes. Numerical simulation of the model enabled us to satisfactorily reproduce the experimental kinetics of formation of BP-phenols, -dihydrodiols, and -tetrols for all tested conditions. Such a validated model was used to investigate the kinetics of unstable genotoxic species such as BP-epoxides and diol epoxides, which were not directly measurable. Based on numerical simulation, BP-7,8-oxide and -9,10-oxide appear to accumulate rapidly to reach a plateau after 2 min, while maximal accumulation of DE2 occurs after about a half-hour and declines during the following 2 h. A contribution of BP-9,10-dihydrodiol metabolism to T2-tetrol formation via the formation of a BP-7,8-oxide-9,10-dihydrodiol is predicted to be detectable after 2 hours due to the preferential accumulation of BP-9,10-dihydrodiol following 1A1-dependent consumption of initially accumulated BP-7,8-dihydrodiol.