CLC proteins form a family of voltage-gated Cl-channels and Cl-/H+-exchangers that are found in all phyla. The human Cl- channels ClC-Ka and ClC-Kb, as their correspondent murine orthologues ClC-K1 and ClC-K2, are almost exclusively expressed in kidney and inner ear epithelia, where they are involved in NaCl reabsorption and endolymph production, respectively. CLC-K channels co-assemble with the β-subunit barttin. Mutations in ClC-Kb and barttin cause Bartter’s syndrome with hypokalemia and salt wasting. Previous reports described CLC-K modulation by extracellular calcium and protons. Currents increase with increasing [Ca2+]ext and are blocked by increasing [H+]ext. An extensive mutagenic screen, based on the crystal structure of the bacterial homologue Ec-CLC-1, combined with voltage clamp measurements on CLC-Ka led us to identify the residue responsible for proton induced block (H497) and two acidic residues responsible for Ca2+ sensitivity (E261 and D278). E261 of one subunit and D278 of the neighboring subunit are close to each other and likely form an intersubunit Ca2+-binding site. All three residues are relatively close, and thus we have identified a novel region involved in the regulation of gating of a CLC channel. To investigate the specificity of the Ca2+ binding site we studied the effect of various divalent cations (Zn2+, Mg2+, Ba2+, Sr2+, Mn2+) on CLC-Ka, CLC-K1, and CLC-Kb. Both WT CLC-Ka and the double mutant E261Q/D278N, were blocked by 5 mM Zn2+ suggesting that Zn2+ affects the channel by binding to a separate binding site. Mg2+ does not activate CLC-Ks at concentrations up to 50 mM. In contrast, CLC-Ka was activated by Ba2+, Sr2+, and Mn2+. The rank order of potency was Ca2+>Ba2+>Sr2+=Mn2+, likely corresponding to a decreasing affinity of these cations. Furthermore, the Ca2+ insensitive double mutant, E261/D278, was also insensitive to Ba2+ and Sr2+ demonstrating the specificity of the mechanism of activation of CLC-K channels by Ca2+. The Ca2+ binding site is conserved in CLC-Kb and CLC-K1 but, interestingly, CLC-K1 showed an altered rank order Ca2+>Sr2+>>Ba>Mg2+. These results will be helpful in the development of a structural model of the cation binding site.