Three isoform-specific antibodies, 6F against the alpha1-isoform of the avian sodium pump, HERED against the rat alpha2-isoform, and Ax2 against the rat alpha3-isoform, were used to detect the expression of Na+-K+-ATPase alpha-subunits in gills of a teleost, the tilapia (Oreochromis mossambicus). Tilapia gill tissue showed positive reactions to antibodies specific for alpha1- and alpha3-isoforms. The results of immunoblots were converted to numerical values (relative intensities) by image analysis for comparisons. Relative amounts of alpha1-like isoform alone and consequently the ratio of alpha1-like to alpha3-like isoforms were higher in gills of seawater-adapted tilapia than in those of freshwater-adapted ones, indicating that the two isoforms respond differently to environmental salinities. In the subsequent immunocytochemical experiments, gill mitochondria-rich cells were demonstrated to immunoreact with antibodies specific for alpha1- and alpha3-isoforms. alpha1-like and alpha3-like isoforms of gill Na+-K+-ATPase are suggested to be involved in the ion- and osmoregulation mechanisms in tilapia. Moreover, differential expressions of two isoforms may be associated with different functions, secretion and uptake of ions and acid-base regulation, in gills of seawater- and freshwater-adapted tilapia.