Reactive oxygen species have been implicated in different types of cardiac arrhythmias including human atrial fibrillation. Kv1.5, the presumed molecular correlate of I_Kur, is an important determinant of human atrial repolarization. The aim of this study was to assess the effects of H₂O₂, at pathophysiologically relevant concentrations (20–1,000 μM), on Kv1.5 expressed in Chinese hamster ovary cell line. Kv1.5 cDNA in pcDNA3 expression vector and CD8, a surface marker protein, were cotransfected in cells by calcium phosphate precipitation. Kv1.5 activation kinetics were significantly accelerated while the activation curve was negatively shifted by 10 mV (V_1/2 changed from −9.3 to −19.0 mV) in the presence of 100 μM H₂O₂. The shift in Kv1.5 peak current I-V curve was voltage-dependent, the current amplitude being increased for voltages <+20 mV but decreased for high depolarizing voltages. The rapid activation time constant obtained from a bi-exponential fitting was decreased from 16.1±3.4 ms to 8.8±1.5 ms for a −20 mV depolarization (n=9; P=0.01) and from 4.3±2.1 ms to 2.3±0.4 ms when cells were depolarized to +20 mV (P<0.05). Kv1.5 steady-state inactivation was not modified by H₂O₂. Intracellular application of SOD or catalase reduced the H₂O₂ induced shift of activation I-V curve and SOD significantly decreased Kv1.5 amplitude at +40 mV (n=9; P<0.05). In conclusion, H₂O₂ increased Kv1.5 current amplitude at voltages corresponding to the action potential repolarization phase and accelerated Kv1.5 channel opening. These changes can reduce the action potential duration, leading to a shortening of the atrial effective refractory period. H₂O₂-induced changes in Kv1.5 properties could thus be involved in initiation or perpetuation of AF.