SUMMARY In the gills of freshwater teleost fishes, vacuolar proton-ATPase (V-H+-ATPase) is found on the apical membrane of pavement and chloride (Na+/K+-ATPase-rich) cells, and is an important transporter for energizing Na+ uptake and H+ excretion. In the gills of elasmobranch fishes, the V-H+-ATPase has not been extensively studied and its expression in freshwater individuals has not been examined. The goals of this study were to examine the effects of environmental salinity on the expression of V-H+-ATPase in the gills of an elasmobranch (the Atlantic stingray, Dasyatis sabina) and determine if V-H+-ATPase and Na+/K+-ATPase are expressed in the same cells. We found that gills from freshwater stingrays had the highest relative abundance of V-H+-ATPase and greatest number of V-H+-ATPase-rich cells, using immunoblotting and immunohistochemistry, respectively. When freshwater animals were acclimated to sea water for 1 week, V-H+-ATPase abundance and the number of V-H+-ATPase-rich cells decreased significantly. Atlantic stingrays from seawater environments were characterized by the lowest expression of V-H+-ATPase and least number of V-H+-ATPase-rich cells. In contrast to teleost fishes, localization of V-H+-ATPase in freshwater stingray gills was not found in pavement cells and occurred on the basolateral membrane in cells that are presumably rich in mitochondria. In freshwater stingrays acclimated to sea water and seawater stingrays, V-H+-ATPase localization appeared qualitatively to be stronger in the cytoplasm, which may suggest the transporter was stored in vesicles. Using a double-immunolabeling technique, we found that V-H+-ATPase and Na+/K+-ATPase occurred in distinct cells, which suggests there may be two types of mitochondrion-rich cells in the elasmobranch gill epithelium. Based on these findings, we propose a unique model of NaCl and acid–base regulation where the V-H+-ATPase-rich cells and Na+/K+-ATPase-rich cells are the sites of Cl– uptake/HCO3– excretion and Na+ uptake/H+ excretion, respectively.