Ventricular arrhythmias commonly originate from the right ventricular out-flow tract (RVOT). However, the electrophysiological characteristics and Ca2+ homoeostasis of RVOT cardiomyocytes remain unclear. Whole-cell patch clamp and indo-1 fluorometric ratio techniques were used to investigate action potentials, Ca2+ homoeostasis and ionic currents in isolated cardiomyocytes from the rabbit RVOT and right ventricular apex (RVA). Conventional microelectrodes were used to record the electrical activity before and after (KN-93, a Ca2+/calmodulin-dependent kinase II inhibitor, or ranolazine, a late sodium current inhibitor) treatment in RVOT and RVA tissue preparations under electrical pacing and ouabain (Na+/K+ ATPase inhibitor) administration. In contrast to RVA cardiomyocytes, RVOT cardiomyocytes were characterized by longer action potential duration measured at 90% and 50% repolarization, larger Ca2+ transients, higher Ca2+ stores, higher late Na+ and transient outward K+ currents, but smaller delayed rectifier K+, L-type Ca2+ currents and Na+-Ca2+ exchanger currents. RVOT cardiomyocytes showed significantly more pacing-induced delayed afterdepolarizations (22% versus 0%, P < 0.05) and ouabain-induced ventricular arrhythmias (94% versus 61%, P < 0.05) than RVA cardiomyocytes. Consistently, it took longer time (9 ± 1 versus 4 ± 1 min., P < 0.05) to eliminate ouabain-induced ventricular arrhythmias after application of KN-93 (but not ranolazine) in the RVOT in comparison with the RVA. These results indicate that RVOT cardiomyocytes have distinct electrophysiological characteristics with longer AP duration and greater Ca2+ content, which could contribute to the high RVOT arrhythmogenic activity.