ABSTRACT Changes in Na+/K+-ATPase activity and abundance associated with environmental salinity were investigated in the gills and rectal gland of the Atlantic stingray Dasyatis sabina. Using a ouabain-specific ATPase assay and western blotting, we found that stingrays from fresh water had the highest activity and highest relative abundance of Na+/K+-ATPase in the gills. Using immunohistochemistry, we also found that gills from freshwater stingrays had the greatest number of Na+/K+-ATPase-rich cells. When freshwater stingrays were acclimated to sea water for 1 week, the activity and abundance of Na+/K+-ATPase and the number of Na+/K+-ATPase-rich cells decreased in the gills. In seawater stingrays, the branchial activity and abundance of Na+/K+-ATPase and the number of Na+/K+-ATPase-rich cells were further reduced. In rectal glands, the activity and abundance of Na+/K+-ATPase were lower in freshwater animals than in seawater-acclimated and seawater stingrays, both of which had equivalent levels. These findings suggest that salinity-associated changes in gill and rectal gland Na+/K+-ATPase activity are due to changes in the abundance of Na+/K+-ATPase. We conclude that the gills may be important for active ion uptake in fresh water, while the rectal gland is important for active NaCl excretion in sea water. The results from this study are the first to demonstrate an effect of environmental salinity on Na+/K+-ATPase expression in the gills and rectal gland of an elasmobranch.