In order to demonstrate the localization of an ethacrynic acid-sensitive Cl- pump in the rat kidney, immunohistochemical analysis was performed using an anti-Cl- pump antibody raised against rat brain Cl- pump protein with confocal laser scanning microscopy. The antibodies against Na+,K+-ATPase, aquaporin 2 and a type B intercalated cell marker, 43-kDa protein, were also used for comparison. Anti-Cl- pump antibody recognized a 51-kDa renal protein of the same size as that in the brain on Western blots. Cl- -pump-like immunoreactivity was observed on the basolateral membranes of 42+/-3% of cortical collecting duct (CCD) cells and of 38+/-1% of outer medullar collecting duct (OMCD) cells. Such immunoreactivity in CCD was sometimes co-localized with Na+,K+-ATPase, but in OMCD, the Cl- pump-like immunoreactivity co-existed with neither Na+,K+-ATPase, aquaporin 2 nor the type B intercalated cell marker 43-kDa protein. Thus, the Cl- pump was demonstrated to be localized on the basolateral membranes of type A intercalated cells of cortical and medullary collecting ducts in the rat kidney.