Using a new sensitive reverse-phase HPLC assay with on-line radioactivity detector, metabolism of (+)-trans-benzo[a]pyrene-7,8-dihydrodiol (B[a]P diol) to the ultimate carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE) was studied using 3-methylcholanthrene-induced rat liver homogenates. The results demonstrate that the stereoselectivity of B[a]PDE formation is a function of the concentration of the cellular constituents in the incubation media. At more dilute concentrations of the homogenate, the ratio of anti- to syn-B[a]PDE was the highest and decreased as the homogenate protein was increased in the incubation medium. However, there was a marked and parallel decrease of free B[a]PDE and DNA-bound radioactivity with increasing concentrations of cellular constituents in the incubation medium. The decreased DNA-bound radioactivity appears to be due to the preferential binding of B[a]PDE to glutathione and to proteins as the homogenate concentration was increased in the incubation media. These results indicate that liver homogenates, while apparently preserving the function of microsomes, present additional opportunities to study the interrelationship among cytochrome P450 monooxygenase activity, water-soluble conjugates, and binding of B[a]P diol metabolites to macromolecules in the study of benzo[a]pyrene-induced carcinogenesis.