Calcium-activated chloride current (ICl(Ca)) is involved in repolarization of the action potential (AP) and its ion channel can be the recently identified TMEM16A. Activation of ICl(Ca) can contribute to the generation of life threatening arrhythmias as delayed afterdepolarizations (DAD) occur especially in calcium-overload but other studies showed ICl(Ca) to be antiarrhythmic.The aim of this work was to evaluate how ICl(Ca) can influence cardiac arrhythmias and to study the regional distribution of ICl(Ca) and TMEM16A.Experiments were performed on isolated canine ventricular myocytes using conventional microelectrode and patch-clamp techniques at 37 C. Adult beagle dogs were anesthetized with I.M. injections of 10 mg/kg ketamine + 1 mg/kg xylazine as approved by the local ethical committee. ICl(Ca) was studied by a selective inhibitor, anthracene-9-carboxylic acid (9-AC, 0.5 mM). Western blot analysis and immunofluorescence of TMEM16A were carried out on cells from various ventricular regions. Values are mean±S.E.M.AP-clamp experiments revealed an early narrow outward and a late inward current peak after the application of 9-AC (+1.15±0.11 pA/pF and -0.18±0.04 pA/pF, respectively, n=13). Similar peak current amplitudes were measured in midmyocardial and subendocardial cells but the early narrow outward current was larger in subepicardial myocytes. Beta-adrenergic activation (10 nM isoproterenol) nearly doubled both peaks of ICl(Ca) suggesting its calcium-sensitivity. AP measurements were in line with this as 9-AC reduced phase-1 magnitude and elevated the mid-plateau potential in a direct rate-dependent manner. 9-AC increased AP duration (APD) at 1 Hz in midmyocardial and subendocardial cells, (+24.34±3.26 ms and +15.85±2.14 ms, n=15 and n=7, respectively) while decreased that in subepicardial myocytes (-13.18±7.50 ms, n=7). Every change of APD was reverse rate-dependent. 9-AC induced APD increase was greater in basal cells (n=13) having longer initial APD compared to that of apical ones (n=15). TMEM16A expression was most abundant in the t-tubules and its density did not differ between apical and basal cells. 9-AC increased the beat-to-beat variability of repolarization and caused early afterdepolarizations (EAD) at low heart rates, especially during beta-adrenergic stimulation.These findings suggest that the different contribution of ICl(Ca) to the AP generation in different cardiac regions is a consequence of the characteristic shape of the given AP. ICl(Ca) equilibrates the apparent transmural and apico-basal temporal difference in repolarization, diminishes the extent of beat-to-beat variability and has a protective role against generation of EAD. Therefore blocking ICl(Ca) could be arrhythmogenic despite it is thought to be beneficial in calcium-overloaded cells by decreasing DADs.