Immunolocalization of Na+,K+-ATPase and morphological changes in gill chloride cells were examined during seawater and freshwater adaptation in the guppy. Immunolocalization was accomplished using a polyclonal antibody specific for the α-subunit of Na+,K+-ATPase. Immunoreactivity appeared in chloride cells on the primary lamellae but differed significantly between two types of the chloride cells, α- and β-chloride cells, in freshwater-adapted guppies. The so-called α-chloride cells, which are elongated cells located at the base of the secondary lamellae, showed strong immunoreactivity whereas the β-chloride cells, which are ovoid cells located at the interlamellar region, showed no or weak immunoreactivity. In the fish of the seawater-adapted strain, most of the chloride cells consisted of the larger α-chloride cells. When freshwater-adapted guppies were transferred to seawater, the α-chloride cells increased in number and size while the β-chloride cells decreased in number. When the fish of the seawater-adapted strain were transferred to freshwater, on the other hand, the α-chloride cells decreased in number and size, whereas the β-chloride cells increased in number. During seawater and freshwater adaptation, the changes in number of the α- and β-chloride cells was accompanied by the appearance of intermediate types between the two types of chloride cells, and the total number of the chloride cells was constant. These results suggest that the α-chloride cells play an important role in active sodium absorption in freshwater and function as the sites of external sodium chloride secretion in seawater, whereas the β-chloride cells have a different role than the α-chloride cells in freshwater and transform themselves into α-chloride cells in seawater. The possible transition of the β-chloride cells into α-chloride cells may be a key point in enabling euryhaline teleosts to adapt to a wide range of salinity environments. J. Exp. Zool. 281:80–89, 1998. © 1998 Wiley-Liss, Inc.