Reactive oxygen species (ROS), including H₂O₂, cause intracellular calcium overload and ischemia-reperfusion damage. The objective of this study was to examine the hypothesis that H₂O₂-induced arrhythmic activity and contractile dysfunction are the results of an effect of H₂O₂ to increase the magnitude of the late sodium current (late I_Na). Guinea pig and rabbit isolated ventricular myocytes were exposed to 200 μM H₂O₂. Transmembrane voltages and currents and twitch shortening were measured using the whole-cell patch-clamp technique and video edge detection, respectively. [Na⁺]_i and [Ca²⁺]_i were determined by fluorescence measurements. H₂O₂ caused a persistent late I_Na that was almost completely inhibited by 10 μM tetrodotoxin (TTX). H₂O₂ prolonged the action potential duration (APD), slowed the relaxation rate of cell contraction, and induced early afterdepolarizations (EADs) and aftercontractions. H₂O₂ also caused increases of [Na⁺]_i and [Ca²⁺]_i. Ranolazine (10 μM), a novel inhibitor of late I_Na, attenuated H₂O₂-induced late I_Na by 51 ± 9%. TTX (2 μM) or 10 μM ranolazine attenuated H₂O₂-induced APD prolongation and suppressed EADs. Ranolazine accelerated the twitch relaxation rate in the presence of H₂O₂ and abolished H₂O₂-induced aftercontractions. Pretreatment of myocytes with ranolazine delayed and reduced the increases of APD, [Na⁺]_i, and [Ca²⁺]_i caused by H₂O₂. In conclusion, the results confirm the hypothesis that an increase in late I_Na during exposure of ventricular myocytes to H₂O₂ contributes to electrical and contractile dysfunction and suggest that inhibition of late I_Na may offer protection against ROS-induced Na⁺ and Ca²⁺ overload.