CLC Proteins comprise 9 members in mammals and have been associated with a variety of physiological functions. Recently, Accardi and Miller have shown that the bacterial CLC protein, which has been crystallized, is a Cl^-/H⁺ antiporter instead of a channel. Some members of the CLC family are clearly channels, e.g., ClC-1 and -2, for which single channel data have been obtained. However, ClC-3, -4, and -5, mainly localized on intracellular vesicles, show a characteristic outward rectification, which has made reversal potential determinations impossible. When we measured pH in ClC-4, or ClC-5-expressing cells using BCECF and clamped these to positive voltages using the gramicidin-perforated patch technique, a strong alkalinization was observed. This alkalinization still occurred when we acidified the extracellular solution such that the driving force would predict acidification through a proton conductance, indicating stoichiometric coupling between Cl^-and H⁺ transport. Depolarizing unpatched cells by the peptide-gated cation channel FaNaC, we showed that alkalinization was dependent on extracellular Cl^-. Alkalinization was not observed when ClC-0 was expressed, or when a critical glutamate was mutated in ClC-4 or -5, conferring linear currents. ClC-3 and -5 have been shown to be essential for synaptic and endocytic vesicle acidification, and are thought to shunt charge accumulated by V-type H⁺-ATPases. Cl^-/H⁺ exchangers dissipate charge, but acidification will require more ATP than with Cl^-channels as shunt.