There is a growing body of evidence that exposure to endocrine disrupting chemicals and to estrogenic compounds in particular can affect the testis and male fertility. In the present study, the constitutive expression of steroidogenic and non-steroidogenic cytochrome P450 (CYP) and related enzymes in adult rat testis, and their regulation by estradiol and bisphenol A, were investigated. CYP1B1, CYP2A1, NADPH-cytochrome P450 oxidoreductase (POR) and microsomal epoxide hydrolase (mEH) proteins, together with CYP17A1 and 3β-hydroxysteroid dehydrogenase (HSD3B), were detected by immunoblot analysis in testicular microsomes prepared from untreated adult Sprague Dawley rats. In contrast, CYP1A, CYP2B, CYP2E, CYP2D, CYP2C, CYP3A, and CYP4A enzymes were not detected. Immunofluorescence staining of cryosections of perfusion-fixed testes showed that CYP1B1, CYP2A1, CYP17A1, and HSD3B were expressed exclusively or mainly in interstitial cells, whereas mEH and POR protein staining was detected both in interstitial cells and in seminiferous tubules. Testicular CYP1B1 and CYP2A1 protein levels were decreased following treatment of adult rats with estradiol benzoate at 0.004, 0.04, 0.4, or 4 μmol/kg/day or bisphenol A at 400 or 800 μmol/kg/day, for 14 days, whereas expression of HSD3B was unaffected. Testicular CYP17A1, POR, and mEH protein expression was also downregulated at the three highest dosages of estradiol benzoate and at both dosages of bisphenol A. The present study is the first to establish the cellular localization of CYP1B1, mEH, and POR in rat testis and to demonstrate the suppressive effect of bisphenol A on testicular CYP1B1, CYP2A1, mEH, and POR protein levels.