Extracellular acidic pH was found to induce an outwardly rectifying Cl^- current (I_Cl,acid) in mouse ventricular cells, with a half-maximal activation at pH 5.9. The current showed a permeability sequence for anions to be SCN^->Br^->I^->Cl^->F^->aspartate, while it exhibited a time-dependent activation at large positive potentials. Similar currents were observed also in mouse atrial cells, and in atrial and ventricular cells from guinea-pig. Some Cl^- channel blockers (DIDS, niflumic acid and glibenclamide) inhibited I_Cl,acid, while tamoxifen had little effect on it. Unlike volume-regulated Cl^- current (I_Cl,vol) and CFTR Cl^- current (I_Cl,CFTR), I_Cl,acid was independent of the presence of intracellular ATP. Activation of I_Cl,acid appeared to be also independent of intracellular Ca²⁺ and G-protein. I_Cl,acid and I_Cl,vol could develop in an additive fashion in acidic hypotonic solutions. Isoprenaline-induced I_Cl,CFTR was inhibited by acidification in a pH-dependent manner in guinea-pig ventricular cells. Our results support the view that I_Cl,acid and I_Cl,vol stem from two distinct populations of anion channels, and that the I_Cl,acid channels are present in cardiac cells. I_Cl,acid may play a role in the control of the action potential duration or cell volume under pathological conditions, such as ischemia-related cardiac acidosis.