Mutations of the human CLCN5 gene, which encodes the CLC-5 Cl^-/H⁺ exchanger, lead to Dent's disease. Mutations result in functional defects that range from moderate reductions to complete loss of whole cell currents, although the severity of functional defect rarely correlates with the severity of the disease. To further elucidate the basis of CLC-5 mutations causing Dent's disease, we examined the functional and cell biological consequences of seven previously reported missense mutants, utilizing electrophysiological and cell biological techniques. This revealed three classes of Dent's disease causing CLC-5 mutations. Class 1 mutations lead to ER-retention and degradation of CLC-5. Class 2 mutations appear to have little effect on subcellular distribution of CLC-5 but cause defective function resulting in severe defects in endosomal acidification. Class 3 mutations lead to alterations in the endosomal distribution of CLC-5 but are otherwise able to support endosomal acidification. Molecular modeling demonstrates a structural basis which may underlie the nature of the defect resulting from each mutation with each class occupying discrete regions of the protein quaternary structure. Thus, these results demonstrate that the cell biological consequences of CLC-5 mutations are heterogeneous, can be classified into 3 major groups and that a correlation between the nature of the defect and the location of the mutation in the structure may be drawn. This model may prove to be useful as a tool to aid in the diagnosis and future therapeutic intervention of the disease.