Real-time optical registration of electrical activity in the heart allows the study of arrhythmogenic mechanisms, in particular due to global ischemia. It is known that global ischemia increases electrical heterogeneity in the heart. However, inter-ventricular differences between the right (RV) and left ventricle (LV) during ischemia and their relationship to arrhythmogenesis remains poorly understood. We used high resolution optical mapping (di-4-ANEPPS, excitation at 532 nm, emission at 640+/-50 nm) of Langendorff-perfused rabbit hearts to quantify inter-ventricular heterogeneity in the heart during periodic pacing and ventricular fibrillation. Two fast CCD cameras were used to record electrical activity from the RV and LV during control, global ischemia (20 min), and reperfusion. Hearts were paced at progressively reduced (from 300 ms to 100 ms) basic cycle lengths and ventricular fibrillation was induced by burst pacing and recorded before the global ischemia, and after the reperfusion. The action potential durations (APD), maximum slopes of APD restitution curves (S(max)), and mean dominant frequency (DF) of ventricular fibrillation were measured for both LV and RV surfaces. No APD heterogeneity was observed in control hearts. Global ischemia induced inter-ventricular heterogeneity in APDs (RV: 109+/-21 ms, LV: 89+/-23 ms; p<0.01) that was abolished upon reperfusion. However, S(max) was uniformly decreased in both RV (control: 0.94+/-0.25, ischemia: 0.36+/-0.12; p<0.01) and LV (control: 0.99+/-0.24, ischemia: 0.43+/-0.21; p<0.01) and did not recover upon reperfusion. In addition, the DF of ventricular fibrillation during reperfusion decreased significantly in RV (from 8.6+/-1.3 Hz to 6.2+/-1.1 Hz; p<0.05) but remained the same in LV (9.0+/-0.8 Hz vs 8.5+/-1.0 Hz). Thus, our results demonstrate that global ischemia induces inter-ventricular heterogeneity in APD during periodic pacing. Although this effect was abolished upon reperfusion, S(max) did not recover, indicating the presence of residual changes in electrical properties of the heart. Therefore, reperfusion reveals the presence of inter-ventricular heterogeneities in the dynamics of ventricular fibrillation.