The current work aimed to apply immunohistochemistry (IHC) to evaluate the differential distribution pattern of some biologically active proteins in the ductus deferens (DD) in two different animal species common in the Egyptian farms. Immunolocalization of S100, angiotensin converting enzyme (ACE), α- smooth muscle actin (α-SMA), connexin 43 (Cx43) and galactosyltransferase (GalTase) was practiced on paraffin-embedded sections of DD from clinically healthy adult five donkeys and five water buffalo bulls. The DD, in both species, was lined by a folded, high cuboidal (in donkey) or tall columnar (in buffalo) pseudostratified epithelium with stereocilia. IHC revealed that all antibodies used, displayed variable reactivities in the two species. Nerves and the periductal smooth muscles expressed a strong immunoreactivity (IR) for S100 and α-SMA respectively; both in the donkey and in the buffalo. Stereocilia of principal cells showed a marked ACE-immunostaining in the donkey but not in the buffalo. Cx43-binding sites were found between the basal portions of the ductal epithelium and the subjacent interstitium; as well as among the periductal smooth muscle cells (SMCs) only in the buffalo bulls. Distinct GalTase-binding sites were found in the ductal epithelium only in the donkey. In conclusion the present findings exhibited a species-specific pattern of distribution for most of proteins under study, suggesting a variable functional significance of the DD in the different animal species.