1,2-Dichlorobenzene 1,4-dichlorobenzene, 1,2,4-trichlorobenzene, 1,2,3,5-tetrachlorobenzene, and pentachlorobenzene were incubated with microsomes derived from cell lines expressing human CYP1A1, CYP1A2, CYP3A4, CYP2E1, or CYP2D6. The formation of phenolic metabolites as determined by gas chromatographic analysis evealed that CYP2E1 possessed the highest activity toward all chlorinated benzenes. Furthermore, CYP1A1 and CYP1A2 showed relatively high enzymatic activities toward the lower chlorinated benzenes (1,2-dichlorobenzene, 1,4-dichlorobenzene, 1,2,4-trichlorobenzene) and CYP3A4 toward the higher chlorinated benzenes (1,2,3,5-tetrachlorobenzene, pentachlorobenzene). CYP2D6 only showed low or nondetectable activity toward the investigated chlorobenzenes. The ratio between the activities of CYP2E1 and CYP3A4 with respect to the oxidation of chlorinated benzenes decreased from 150 (1,2-dichlorobenzene) to 1.8 (pentachlorobenzene). In order to estimate the relative contribution of CYP2E1 in hepatic metabolism of 1,2-dichlorobenzene and 1,2,4-trichlorobenzene in vitro, the rate of oxidation of these compounds by microsomal preparations from 22 human livers was correlated with activities toward specific substrates for CYP2E1, CYP3A, and TYP1A. The results were supportive for the results obtained with single human P450 enzymes. CYP2E1 is the major, if not the only, enzyme involved in the formation of 2,3-dichlorophenol, 3,4-dichlorophenol, 2,3,5-trichlorophenol, and 2,3,4-trichlorophenol, while CYP3A4 is responsible for the formation of 2,3,6-trichlorophenol. The formation of the major metabolite from 1,2,4-trichlorobenzene (2,4,5-trichlorophenol) was correlated with both CYP2E1 and CYP3A activity. Because of the decreasing ratio in activity between CYP2E1 and CYP3A4 with respect to the oxidation of chlorinated benzenes, it is concluded that the role of CYP2E1 toward chlorinated benzenes decreases with increasing number of chlorine atoms. The relative amount of CYP3A4 present then becomes an important determinant for metabolism.