Previous studies indicate that the colonic H-K-ATPase mRNA is expressed as the distal nephron. However, the exact intrarenal localization of the colonic H-K-ATPase protein is still unclear. The goal of the present study was to determine the cellular and subcellular localization of the colonic H-K-ATPase protein in the rabbit kidney. We used three monoclonal antibodies (MAbs) directed against different epitopes of the rabbit colonic H-K-ATPase α-subunit (HKα2) to localize HKα2protein by immunofluorescence labeling of kidney sections and laser-scanning confocal microscopy. The specificity of the MAbs was confirmed by reaction with a single ∼100-kDa band on Western blots of distal colon. Specific immunohistochemical reaction with the apical membrane of surface epithelial cells was observed with all three MAbs on distal colon sections. In rabbit kidney, immunofluorescence was detected only on the apical membrane of connecting tubule cells. Immunofluorescence was not detected in the cortical-, outer-, and inner-medullary collecting ducts. Furthermore, costaining with principal- and intercalated cell-specific MAbs and a MAb against the thick ascending limb suggests that these cell types express HKα2protein at levels that are below the detection limit with this method. We conclude that in the rabbit kidney, under normal dietary conditions, the HKα2protein is expressed in the apical membrane of connecting tubule cells.