The CLCA family of Ca2+-activated Cl- channels has recently been discovered, with an increasing number of closely related members isolated from different species. Here we report the cloning of the second human homolog, hCLCA2, from a human lung cDNA library. Northern blot and RT-PCR analyses revealed additional expression in trachea and mammary gland. A primary translation product of 120 kDa was cleaved into two cell surface-associated glycoproteins of 86 and 34 kDa in transfected HEK-293 cells. hCLCA2 is the first CLCA homolog for which the transmembrane structure has been systematically studied. Glycosylation site scanning and protease protection assays revealed five transmembrane domains with a large, cysteine-rich, amino-terminal extracellular domain. Whole cell patch-clamp recordings of hCLCA2-transfected HEK-293 cells detected a slightly outwardly rectifying anion conductance that was increased in the presence of the Ca2+ ionophore ionomycin and inhibited by DIDS, dithiothreitol, niflumic acid, and tamoxifen. Expression in human trachea and lung suggests that hCLCA2 may play a role in the complex pathogenesis of cystic fibrosis.