Na,K-ATPase (sodium pumps) provide the primitive driving force for ion transport in branchial epithelial cells. Immunoblots of epithelial homogenates of both seawater (SW)- and freshwater (FW)-adapted tilapia gills as well as rat brain homogenate, a positive control, revealed one major band with a molecular weight of about 100 kDa. SW-adapted tilapia gills possessed larger (about 2-fold) amounts of sodium pumps compared with FW-adapted tilapia gills. (3)H-ouabain binding representing functional binding sites of Na,K-ATPase was also higher (about 3.5-fold) in gills of SW-adapted tilapia compared to that of FW-adapted fish. Moreover, specific activities of SW fish were higher (about 2-fold) than those of FW fish. Double labeling of Na,K-ATPase and Con-A, a fluorescent marker of MR cells, in tilapia gills followed by analysis with confocal microscopy showed that sodium pumps were localized mainly in MR cells, including the SW type and different FW types. Although more-active expression of Na,K-ATPase was demonstrated in gills of SW-adapted tilapia, no significant differences in densities of apical openings of MR cells were found between SW- and FW-adapted fish. These results indicate that, during salinity challenge, tilapia develop more "functional" Na,K-ATPase in SW-type MR cells to meet physiological demands.