CLC-5 is a member of the family of voltage-gated Cl^– channels and Cl^–/H⁺ antiporters [1,2]. It is expressed in the proximal tubule of the kidney where it plays a role in the reabsorption of albumin from the glomerular filtrate. CLC-5 is mainly localised to endosomal membranes and has been proposed to provide a conductance through which Cl^– ions can pass into the vesicle lumen to counterbalance the influx of H⁺ pumped by H⁺-ATPase. Mutations of ClC-5 have been linked to Dent’s disease, an X-linked disorder characterised by proteinurea, hypercalciurea, and nephrolithiasis [3]. Dent’s mutants have previously been described as those that completely ablate CLC-5 currents or those that give either increased or decreased currents. However, the role of surface membrane CLC-5 in the proximal tubule is unclear and nothing is known about the properties of mutant CLC-5 in organelles, where it is though to play a key physiological role. We therefore examined both the electrophysiology and cell biology of several known disease-causing missense mutations in an attempt to further our understanding of the precise CLC-5 defect in Dent’s disease. Wild type and mutant EYFP-CLC-5 were transiently transfected into HEK-293 cells and studied by patch clamp electrophysiology, confocal imaging, and protein biochemistry. Whole cell currents were recorded from fluorescing cells using CsCl-based solutions, holding the cell at -30mV and applying 10ms pulses from -100 to +200mV. Compared with wild-type CLC-5, two of the mutations, G57V and R280P, reduced the current amplitude whilst no significant whole-cell currents were recorded from cells expressing the mutants S270R, G513E, R516W, I524K, and E527D. These properties correlated with the sub-cellular distribution of the wild-type and mutant EYFP-CLC-5: mutations that reduced whole-cell currents were predominantly located in intracellular endosomes and to varying extents at the cell surface whilst mutants that abolished whole-cell currents displayed a diffuse pattern of staining consistent with ER-localisation and retention. One exception was E527D which did not pass current when examined by electrophysiology, but was located predominantly in punctuate endosomal structures. All of these mutations result in proteinurea in Dent’s patients, but there exists a functional heterogeneity of disease-causing mutations that correspond to a structure-function relationship. Further studies are required to determine the physiology of wild-type and mutant CLC-5 in both surface and organelle membranes to understand their role in normal and diseased kidney function.