The ionic nature of endosomes varies considerably in character along the endocytic pathway. Counter-ion flux across the limiting membrane of endosomes has long been considered essential for full acidification and normal endosome/lysosomal function. The proximal functions of luminal ions, however, have been difficult to assess. The recent development of transgenic mice carrying mutations in the intracellular chloride channels (ClCs) has provided a tool to uncouple Cl− influx from endosomal acidification. Intriguingly, many of the defects of the endo-lysomal system attributed to aberrant pH persist in the Cl−-deficient mice implying a direct regulatory role for Cl− influx in endosome function. These observations may begin to explain the abundance of endosomal ion transporters, including ClCs, sodium-proton exchangers, two-pore channels and mucolipins, that have been localized to endo-lysosomes, and the extensive changes in luminal ion composition therein. In this review, we summarize what is known regarding the mediators of endosomal ion flux, and discuss the implications of changing ionic content on endo-lysosomal function.