Endosomes maintain a slightly acidic internal pH, which is directly responsible for their ability to ensure proper sorting of incoming receptors and ligands during endocytosis. At least two distinct subpopulations of endosomes can be distinguished, designated "early" and "late" on the basis of their kinetics of labeling with endocytic tracers. The subpopulations differ not only in their functions (rapid receptor recycling and transport to lysosomes, respectively) but also in their capacities for acidification in intact cells and in vitro. To investigate the possible basis for pH regulation in endosomes, we have studied the transport properties and ion permeabilities of early and late endosomes isolated from Chinese hamster ovary cells. Using endosomes selectively labeled with pH-sensitive endocytic tracers, we found that ATP-dependent acidification is electrogenic, being accompanied by the generation of an interior-positive membrane potential which opposes further acidification. While membrane potential and, consequently, acidification was controlled by the influx of permeant anions and efflux of protons and alkali cations, acidification was further modulated in Na+ and K+-containing buffers by the ouabain- and vanadate-sensitive Na+,K+-ATPase, which appears to be a functional component of the endosomal membrane. The data suggest that electrogenic Na+ transport due to Na+,K+-ATPase activity contributes to the interior-positive membrane potential, thereby reducing ATP-dependent H+ transport. Importantly, inhibition of acidification by Na+,K+-ATPase activity was found only in early endosomes, consistent with their limited acidification capacity relative to late endosomes and lysosomes.