A volume-regulated chloride current ( I_Cl.vol) is ubiquitously present in mammalian cells, and is required for the regulation of electrical activity, cell volume, intracellular pH, immunological responses, cell proliferation and differentiation. However, the molecule responsible for I_Cl.vol has yet to be determined^,,. Although three putative chloride channel proteins expressed from cloned genes (P-glycoprotein, p I_Cln and ClC-2 ) have been proposed to be the molecular equivalent of I_Cl.vol, neither P-glycoprotein nor p I_Cln is thought to be a chloride channel or part thereof,, and the properties of expressed ClC-2 channels differ from native I_Cl.vol (refs. , ). Here we report that functional expression in NIH/3T3 cells of a cardiac clone of another member of the ClC family, ClC-3, results in a large basally active chloride conductance, which is strongly modulated by cell volume and exhibits many properties identical to those of I_Cl.vol in native cells^,,,^,,,,. A mutation of asparagine to lysine at position 579 at the end of the transmembrane domains of ClC-3 abolishes the outward rectification and changes the anion selectivity from I⁻ > Cl⁻ to Cl⁻ > I⁻ but leaves swelling activation intact. Because ClC-3 is a channel protein belonging to a large gene family of chloride channels,, these results indicate that ClC-3 encodes I_Cl.vol in many native mammalian cells.