The present study was designed to evaluate the putative expression of K_ATP channels subunits in epididymis from various species by means of immunohistochemical and Western blot techniques. By using the ABC-DAB approach, a positive staining for Kir6.2 and SUR2 was observed in the principal epithelial cells throughout all regions of the rat epididymis (initial segment, caput, corpus, cauda). At higher magnification, the Kir6.2 staining exhibited a supranuclear whorl pattern whilst the SUR2 labelling was diffusely cytoplasmic. Immunofluorescent detection confirmed the co-localisation of Kir6.2 and SUR2 in principal cells of rat epididymal epithelium. Confocal microscopy further ascertained the whorl supranuclear staining of Kir6.2 and the more diffuse cytoplasmic SUR2 staining. Rat and mouse epididymal spermatozoa also stained for Kir6.2 and SUR2. Western blot analysis of epididymal protein extracts from adult and prepubertal rats revealed, for Kir6.2, a double band at 38kDa and a single band below 29kDa. Immunoblot failed to detect SUR2 expression. Lastly, a staining for Kir6.2 and SUR2 was also detected in human, canine, feline and bovine epididymal specimens. These data demonstrate the presence of ATP-sensitive K⁺ channels subunits, Kir6.2 and SUR2, in epididymal epithelial cells from various species. The physiological roles of epididymal ATP-sensitive K⁺ channels need to be further elucidated.