Expression of estrogen receptors (ERs) in the reproductive tracts of adult male dogs and cats has not been reported. In the present study, ERα and ERβ were localized by immunohistochemistry using ER-specific antibodies. ERα was found in interstitial cells and peritubular myoid cells in the dog testis, but only in interstitial cells of the cat. In rete testis of the dog, epithelial cells were positive for ERα staining, but in the cat, rete testis epithelium was only weakly positive. In efferent ductules of the dog, both ciliated and nonciliated cells stained intensely positive. In the cat, ciliated epithelial cells were less stained than nonciliated epithelial cells. Epithelial cells in dog epididymis and vas deferens were negative for ERα. In the cat, except for the initial region of caput epididymis, ERα staining was positive in the epithelial cells of epididymis and vas deferens. Multiple cell types of dog and cat testes stained positive for ERβ. In rete testis and efferent ductules, epithelial cells were weakly positive for ERβ. Most epithelial cells of the epididymis and vas deferens exhibited a strong positive staining in both species. In addition, double staining was used to demonstrate colocalization of both ERα and ERβ in efferent ductules of both species. The specificity of antibodies was demonstrated by Western blot analysis. This study reveals a differential localization of ERα and ERβ in male dog and cat reproductive tracts, demonstrating more intensive expression of ERβ than ERα. However, as in other species, the efferent ductules remained the region of highest concentration of ERα.