Recently, a novel type of anion channel activated by extracellular acidification has been found in a variety of mammalian cell types. However, the role of this acid-sensitive outwardly rectifying (ASOR) anion channel is not known. In human epithelial HeLa cells, reduction in extracellular pH below 5 rapidly activated anion-selective whole-cell currents. The currents exhibited strong outward rectification, activation kinetics at positive potentials, low-field anion selectivity, and sensitivity to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and phloretin. When outside-out patches were exposed to acidic bathing solution, single-channel events of the anion channel could be observed. The unitary conductance was 4.8 pS in the voltage range between -80 and +80 mV. The single-channel activity prominently increased with depolarization and was suppressed by DIDS or phloretin. After 1-h incubation in acidic solution (pH 4.5), a significant population of HeLa cells suffered from necrotic cell injury characterized by stainability with propidium iodide and lack of caspase-3 activation. Upon exposure to acidic solution, HeLa cells exhibited immediate, persistent swelling. Both the necrotic volume increase and cell injury induced by extracellular acidification were inhibited by DIDS or phloretin. Therefore, it is concluded that the ASOR anion channel is involved in the genesis of necrotic cell injury induced by acidosis in human epithelial cells.