In ventricular myocytes, key proteins involved in excitation-contraction coupling are located predominantly at the t-tubules (TT) membrane. While TT have also been described in atrial myocytes of large species, information regarding Ca2+ cycling and TT network in different regions of left atrium (LA) are lacking. Knowing that LA plays a prominent role in atrial fibrillation, we have examined Ca2+ transients (CaTr) and TT characteristics in 4 different LA regions in an animal model that translates to human. Procedures were in accordance with guidelines from Directive 2010/63/EU of the European Parliament. Sheep (N=20, 50±2 Kg) were pre-medicated with 20 mg/kg ketamine and 10-20 mg/kg acepromazine (both i.m.). Anesthesia was induced with sodium pentobarbital (10 mg/kg, i.v.) and maintained with 2-3% isoflurane. Heparin (200 IU/Kg, i.v.) was injected and sheep were euthanized with pentobarbital (20 mL, i.v.). Heart was excised and rinsed with cardioplegic solution. LA was cut, cannulated via the circumflex artery and perfused 20 min with 0.1 mM EGTA isolation solution (IS), followed by ~30 min perfusion with IS containing 1 mg/mL type II collagenase (Worthington), 0.1 mg/mL type XIV protease (Sigma) and 0.08 mM Ca2+. Enzymes were washed out with 0.2 mM Ca2+ IS. Myocytes were obtained from epicardial (EPI) and endocardial (Endo) surface of left appendage, free wall (FW) and pulmonary veins sleeves (PV). CaTr was recorded (Fura-2, 0.5Hz) using an IonOptix system. Cell membrane was stained with di-8 ANNEPS and visualized under confocal microscopy. Values are mean±S.E.M. compared by ANOVA. No difference were observed for diastolic Ca2+ nor CaTr amplitude (diastolic Ca2+: EPI= 0.95±0.01 Ratio Unit, [RU] (n=24), Endo= 0.93±0.01 RU (n=26), FW= 0.91±0.01 RU (n=30) and PV= 0.92±0.01 RU (n=22); NS; CaTr: Epi= 0.013±0.002 RU, Endo= 0.013±0.002 RU, FW= 0.015±0.002 RU and PV= 0.016± 0.002 RU; NS). Compared to left appendage, time to peak (TTP) and time to 50% decay (TTD) were longer in the 3 other regions (TTP in ms: 142±10 Endo, 211±22 FW and 198±28 PV vs 97±8 EPI; P<0.05; TTD in ms: 509±23 Endo, 676±21 FW and 640±20 PV vs 380±22 EPI, P<0.05). Longest TTD was observed in FW and PV cells; P<0.05 compared to Endo. TT were present in all cell types albeit the percentage of cells displaying organized TT network varied (% of cells expressing TT: 75% Epi (n=81), 85% Endo (n=79), 63% FW (n=59) and 57% PV (n=76)). TT density (in µm-1) was significantly lower in PV and FW compared to Epi and Endo cells (0.021±0.001 Epi (n=48), 0.019±0.001 Endo (n=63), 0.014±0.001 FW (n=43), 0.012±0.001 PV (n=65), P<0.05). LA displays region-specific kinetics of Ca2+ signaling. These data are consistent with TT density observed. Overall, those results suggest regional differences in CaTr and TT in LA may play a major role in AF genesis.