Furosemide administration stimulates distal acidification. This has been attributed to the increased lumen-negative voltage in the distal nephron, but the aspect of regulatory mechanisms of H⁺-ATPase has not been clear. The purpose of this study is to investigate whether chronic administration of diuretics alters the expression of H⁺-ATPase and whether electrogenic Na⁺ reabsorption is involved in this process. A seven day infusion of furosemide or hydrochlorothiazide (HCTZ) lowered urine pH significantly. However, this effect of furosemide-induced distal acidification was not changed with amiloride blocking electrogenic Na+ reabsorption. On immunoblots, a polyclonal antibody against the H⁺-ATPase B1 subunit recognized a specific ~56-kDa band in membrane fractions from the kidney. The protein abundance of H⁺-ATPase was significantly increased by furosemide and HCTZ infusion both in the cortex and outer medulla. Furosemide plus amiloride administration also increased the H⁺-ATPase protein abundance significantly. However, no definite subcellular redistribution of H⁺-ATPase was observed by furosemide ± amiloride infusion in the immunohistochemistry. Chronic furosemide ± amiloride administration induced a translocation of pendrin to the apical membrane, while total protein abundance was not increased. The mRNA expression of H⁺-ATPase was not altered by furosemide ± amiloride infusion. We conclude that chronic administration of diuretics enhances distal acidification by increasing the abundance of H⁺-ATPase irrespective of electrogenic Na⁺ reabsorption. This upregulation of H⁺-ATPase in the intercalated cells may be the result of tubular hypertrophy by diuretics.