ClC-5 is a member of the mammalian family of voltage-dependent Cl^– channels that acts as a Cl^–/H⁺ exchanger (Picollo & Pusch, 2005; Scheel et al. 2005). Inherited mutations of CLCN5 lead to defective albumin reabsorption in the proximal tubule (Dent’s disease). At the cellular level, ClC-5 co-localises with v-ATPase in endosomes of the proximal tubule epithelial cells, where it is thought to provide a Cl^– conductance to counterbalance the active transport of H⁺ into the endosomal lumen (Günther et al. 1998). Whether the physiological properties of ClC-5 permit this shunting role has yet to be fully addressed. We transfected human embryonic kidney cells (HEK 293) with plasmid DNA encoding EYFP-tagged ClC-5 and recorded currents using the whole-cell patch clamp technique. The bath solution contained (in mM) 140 CsCl, 1 CaCl₂, 1 MgCl₂, and 10 HEPES, and the pipette solution contained 42 CsCl, 98 aspartate, 10 EGTA, and 10 HEPES; both solutions adjusted to pH 7.4. Cells were held at -30mV and 10ms pulses were applied from -100 to +200mV. Cells expressing ClC-5-EYFP produced outwardly-rectifying currents with a half-maximal activation voltage of +110 ± 2mV and an equivalent gating charge of 1.32 ± 0.13 (mean ± s.e.m., n=5). These currents, when extrapolated to the endosomal membrane, correspond to a unidirectional flux of chloride from the lumen to the cytosol. Currents were measured with [Cl^–] ranging from 10 to 140mM and pH from 8.4 to 5.4 on both sides of the membrane, but on no occasion were inward currents observed. To further understand the physiological role of ClC-5 in endosomal membranes we studied acidification using a pH-sensitive fluorescent protein, pHluorin, targeted to endosomes by fusion to VAMP2 (Miesenbock et al., 1998). In an extracellular buffer with pH 7.4, untransfected HEK cells acidified endosomal contents to pH 6.98 ± 0.05 (n=25), but failed to acidify endosomes (pH 7.40 ± 0.08, n=15) in the presence of 25μM bafilomycin, a v-ATPase inhibitor. HEK cells expressing ClC-5 had endosomal pH 6.41 ± 0.18 (n=30) and in the presence of bafilomycin, pH 6.87 ± 0.18 (n=16), indicating a bafilomycin-insensitive acidification mechanism when ClC-5 is present. The data suggest that, in endosomes, ClC-5 does not act as en electrical shunt, but conducts Cl^– ions from the lumen to the cytosol. Since this is coupled to an exchange for H+ this may provide an acidification mechanism that acts in parallel with vesicular v-ATPase. This enhanced acidification in early endocytic vesicles may be important for efficient albumin endocytosis in the proximal tubule.