The action of glucocorticoids in target tissues is dependent on the local expression of glucocorticoid receptors and two 11β-hydroxysteroid dehydrogenase (11β-HSD) enzymes, 11β-HSD1 and 11β-HSD2, which interconvert active and inactive glucocorticoids. This study examined expression of the 11β-HSD enzymes in the male reproductive tract of the adult rat. 11β-HSD1 was immunolocalized to the apical region of principal epithelial cells of the caput epididymis, with the less numerous clear cells devoid of signal. Epididymal 11β-HSD1 expression was confirmed by Western blot analysis, with immunoreactive species identified at 34 kDa (the expected size for 11β-HSD1) and at approximately 48 kDa. 11β-HSD bioactivity was readily detectable in the epididymis, with 11-oxoreductase activity clearly the favored reaction (as observed in liver), consistent with 11β-HSD1 expression. The epithelium of the vas deferens, seminal vesicle, and penile urethra were also immunopositive for 11β-HSD1, as were smooth muscle cells of the vas deferens and penile blood vessels. 11β-HSD2 was also immunolocalized to the epididymal epithelium, but its distribution was complementary to that of 11β-HSD1 (i.e. clear cells showing intense 11β-HSD2 staining but principal cells devoid of signal). 11β-HSD2 was also present in the corpora cavernosa of the penis but not in other tissues. In conclusion, the differential expression of 11β-HSD1 and 11β-HSD2 throughout the male reproductive tract suggests that these enzymes locally modulate glucocorticoid and mineralocorticoid actions, particularly in the epididymis and penile vasculature.