Kidney proximal tubule epithelial cells have an extensive apical endocytotic apparatus that is critical for the reabsorption and degradation of proteins that traverse the glomerular filtration barrier and that is also involved in the extensive recycling of functionally important apical plasma membrane transporters. This process involves the passage of apical recycling proteins through acidic endosomes that contain the vacuolar H⁺-ATPase. We have previously shown that the expression of some cell-surface proteins in the proximal tubule is decreased in cadmium nephrotoxicity, and that cadmium inhibits endosomal acidification. Furthermore, cadmium toxicity induces a Fanconi syndrome characterized by low molecular weight proteinuria, glucosuria, aminoaciduria, etc. Furthermore, Dent’s disease, a hereditary disease caused by a mutation in the CLC5 chloride channel, also causes a Fanconi-like syndrome associated with hypercalcuria and kidney stone formation. It is postulated that the chloride channel mutation results in defective endosomal acidification in proximal tubules, leading to loss of apical transport proteins and Fanconi syndrome. Previous studies had shown that intra-vesicular acidification is required for coat protein recruitment to vesicles and our present work was aimed at further dissecting the mechanism by which aberrant endosomal acidification might cause functional defects in the apical recycling pathway of the proximal tubule. First, we localized the Arf GTP/GDP exchange factor, ARNO (ADP-Ribosylation factor Nucleotide site Opener) as well as Arf6 and Arf1 small GTPases in the kidney proximal tubule receptor-mediated endocytosis pathway. In proximal tubules in situ, ARNO and Arf6 were found in the apical pole of epithelial cells associated with endosomes and partially colocalized with the vacuolar H⁺-ATPase. Arf1 was localized both in the Golgi and at the apical pole of proximal tubule epithelial cells. Western blot analysis showed that ARNO, Arf6 and Arf1 were also present both in purified endosomes and cytosol of proximal tubules. Therefore, we examined the cytosol-to-endosomal membrane recruitment of endogenous ARNO and Arf small GTPases in vitro. Using purified proximal tubule endosomes in a translocation assay, we found that ATP-driven endosomal acidification triggers the recruitment of ARNO from cytosol to endosomal membranes. This recruitment of ARNO was also accompanied by selective pH-dependent translocation of Arf6 but not Arf1 from cytosol to endosomal membranes. The translocation of both ARNO and Arf6 was reversed by V-type ATPase inhibitors as well as by uncouplers of endosomal intraluminal pH, and was correlated with the magnitude of intra-endosomal acidification. Our data suggest that V-H⁺-ATPase-dependent acidification may be responsible for the selective recruitment of ARNO and Arf6 to proximal tubule early endosomes in situ and therefore may play an important role in regulating the receptor-mediated endocytosis pathway. This would provide a potential link between defective endosomal acidification, aberrant apical membrane protein recycling, and the functional defects that are associated with a loss of apical transporters from the proximal tubule in both acquired (e.g. cadmium nephrotoxicity) and hereditary (e.g. Dent’s disease) disorders that lead to renal Fanconi syndrome.