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(1-3). Although three putative chloride channel proteins expressed from cloned genes (P-glycoprotein(4), pI(Cln), (ref. 5) and ClC-2 (ref. 6)) have been proposed to be the molecular equivalent of I-Cl.vol, neither P-glycoprotein nor pI(Cln), is thought to be a chloride channel or part thereof(7,8), and the properties of expressed ClC-2 channels differ from native I-Cl.vol (refs. 3, 6). Here we report that functional expression in NIH/3T3 cells of a cardiac clone of another member of the CIC 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(1-3,9-13). 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(3,14), these results indicate that ClC-3 encodes I-Cl.vol in many native mammalian cells.