The proper functioning of the Cl^- channel, ClC-5, is essential for the uptake of low-molecular weight proteins through receptor-mediated endocytosis in the proximal tubule. Dent's disease patients with mutant ClC-5 channels and ClC-5 knockout (KO) mice both have low molecular weight proteinuria. To further understand the function of ClC-5, endocytosis was studied in LLC-PK1 cells and primary cultures of proximal tubule cells from wild type (WT) and ClC-5 knockout (KO) kidneys. Endocytosis in the proximal tubule cells from KO mice was reduced compared to that in WT animals. Endocytosis in WT but not in KO cells was inhibited by Bafilomycin A-1 and Cl^- depletion, whereas, endocytosis in both WT and KO cells was inhibited by the NHE3 blocker, S3226. Infection with adenovirus containing WT ClC-5 rescued receptor-mediated endocytosis in KO cells, whereas, infection with any of three disease causing mutants, myc-W22G-ClC-5, myc-S520P-ClC-5 or myc-R704X-ClC-5 did not. WT and the three mutants all trafficked to the apical surface, as assessed by surface biotinylation. WT-ClC-5 and the W22G mutant were internalized similarly, while neither the S520P nor the R704X mutants was. These data indicate that ClC-5 is important for Cl^- and proton pump mediated endocytosis. However, not all receptor-mediated endocytosis in the proximal tubule is dependent upon ClC-5. There is a significant fraction which can be inhibited by an NHE3 blocker. Our data from the mutants suggest that defective targeting and trafficking of mutant ClC-5 to the endosomes is a major determinant in the lack of normal endocytosis in Dent's Disease.