Loss of the endosomal anion transport protein ClC-5 impairs renal endocytosis and underlies human Dent's disease. ClC-5 is thought to promote endocytosis by facilitating endosomal acidification through the neutralization of proton pump currents. However, ClC-5 is a 2 chloride (Cl⁻⁾/proton ⁽/H⁺) exchanger rather than a Cl⁻ channel. We generated mice that carry the uncoupling E211A (unc) mutation that converts ClC-5 into a pure Cl⁻ conductor. Adenosine triphosphate (ATP)-dependent acidification of renal endosomes was reduced in mice in which ClC-5 was knocked out, but normal in Clcn5unc mice. However, their proximal tubular endocytosis was also impaired. Thus, endosomal chloride concentration, which is raised by ClC-5 in exchange for protons accumulated by the H⁺-ATPase, may play a role in endocytosis.