ClC-5 is a member of the voltage-gated chloride channel family that acts as a Cl^–/H⁺ antiporter [1,2]. It is expressed in the proximal tubule of the kidney where it involved in small-peptide reabsorption from the urine. ClC-5 is predominantly located on endosomal membranes and has been proposed to provide a Cl^– conductance to counterbalance the action of v-ATPase. Mutation of ClC-5 has been linked to Dent’s disease, an X-linked disorder characterised by proteinurea, hypercalciurea and nephrolithiasis [3]. We have examined the functional consequences of several known Dent’s disease missense mutations. Data are expressed as mean ± s.e.m., statistical significance determined by ANOVA. Following expression of wild-type (WT) and mutant EYFP-tagged ClC-5 in HEK293 cells, whole-cell currents were examined by patch-clamp electrophysiology. Current densities of cells expressing the G57V mutant (269±30pA/pF) were not different to WT (319±45pA/pF) (p>0.05, n=5) whereas R280P exhibited currents that were reduced by ~50% (155±46pA/pF) (p<0.05; n=5). Whole-cell currents of 5 other mutants (S270R, G513E, R516W, I524K & E527D) were not different to untransfected cells (p<0.05; n≥5). Confocal imaging of cells showed that WT, G57V, R280P and E527D were located predominantly in intracellular endosomes and to varying extents at the cell surface. The four remaining mutants were retained in the endoplasmic reticulum. Endocytosis was examined in cells expressing WT and endosome-targeted mutant ClC-5. The uptake of fluorescently-conjugated albumin was increased ~5-fold in cells expressing WT, G57V or R280P versus untransfected (p<0.01; n≥6) but not in E527D expressing cells (p>0.05; n=11). Uptake of fluorescently-conjugated dextran and transferrin were not affected by the presence of ClC-5 (p>0.05 vs untransfected; n≥4). Endosomal acidification was assayed using ratiometric pHluorin fused to VAMP2 for endosomal targeting [4]. Endosomes of untransfected cells bathed in pH 7.4 buffer were acidified to pH 6.98 ± 0.05. Significant acidification was observed in cells expressing WT (pH 6.40 ± 0.17), G57V (pH 6.25 ± 0.19) and R280P (pH 5.46 ± 0.15) (p<0.01 vs untransfected; n≥15 cells for each). Acidification in cells expressing E527D was impaired leading to a modest alkalisation compared to untransfected cells (pH 7.55 ± 0.09) (p<0.05; n=15 cells). These data suggest that Dent’s disease does not necessarily result from a loss of a ClC-5 conductance from the endosome, but could be caused by disruption of another unidentified process involving ClC-5. They also suggest that efficient albumin endocytosis requires functional ClC-5 at both surface and endosomal membranes.