The electrophysiologic effects of hydrogen peroxide on the isolated guinea pig right ventricular free wall were studied using simultaneous recordings of action potentials from the epicardium and the endocardium. Exposure to hydrogen peroxide caused a time- and concentration-dependent change in action potential characteristics. Action potential durations at 50 and 90% of repolarization (APD50 and APD90, respectively) were significantly prolonged by hydrogen peroxide in both the epicardium and the endocardium. Although prolongation occurred at lower concentrations (0.5 mM) in the epicardium, increases in APD in response to higher concentrations of hydrogen peroxide (1 or 4 mM) were maintained for a longer period of time in the endocardium. In addition, hydrogen peroxide (1 or 4 mM) caused significant depolarization in the epicardium after 10 min, although this effect was observed only in the endocardium exposed to 4 mM hydrogen peroxide. Ventricular arrhythmias were observed in 5 of 7, 6 of 7, and 7 of 7 preparations exposed to 0.5, 1, and 4 mM hydrogen peroxide, respectively. The most frequently observed electrophysiologic abnormalities were associated with increased automaticity. Coupled beats, including clearly indentifiable early and delayed depolarizations, were also observed. Verapamil (2 μM) and amiloride (0.1 mM) reduced both the incidence and the duration of hydrogen peroxide-induced arrhythmias but did not influence the effects on APD. This study is the first demonstration of hydrogen peroxide-mediated transmural dispersion in APD that could play an important role in the development of ventricular arrhythmias. In addition, our results demonstrate that hydrogen peroxide can induce ventricular arrhythmias through several cellular mechanisms, including increased automaticity, coupled beats, and triggered activity.