Cytochrome P450 2E1 participates in the bioactivation of a wide variety of environmental and occupational pollutants. Such reactions may lead to the production of active carcinogenic metabolites. The presence of P450 2E1 in the testis and prostate has not yet been reported. In the present study, cytochrome P450 2E1 mRNA has been identified in the rat prostate and testis by reverse transcription PCR, southern blotting, and DNA sequencing. P450 2E1 protein from rat testis could be detected with immunoblot analysis, but was not detected in the prostate. The hydroxylation ofp-nitrophenol, known to be mediated by P450 2E1, was demonstrated by HPLC measurement of product formation in microsomal fractions from the rat testis, but again not from prostate. Exposure of rats to pyridine resulted in a 2.9-fold increase ofp-nitrophenol hydroxylation by testicular microsomes. Diethyldithiocarbamate, a selective mechanism-based inhibitor of P450 2E1, or a P450 2E1 monoclonal antibody, caused marked inhibition of testicular microsomalp-nitrophenol hydroxylase activity. These results indicate that cytochrome P450 2E1 is present in the rat testis, and that it is elevated by treatment of the animals with pyridine. Thus, the presence and inducibility of cytochrome P450 2E1 in the testis may be of significance in the bioactivation of environmental chemicals to genotoxic metabolites.