Action potential alternans has been an indicator of ischemic disease and vulnerability to ventricular fibrillation (VF). The mechanisms of alternans are linked to the anomalies in intracellular Ca2+ (Cai) handling by either spontaneous Ca2+ release or modulation of action potential duration (APD), which may promote wave breaks in VF. We investigated possible role of Ca2+ in wave breaks by simultaneously measuring transmembrane potential (Vm) and intracellular Ca2+ concentration with voltage sensitive dye (RH237) and Ca2+ (Rhod-2) fluorescence probes. VF was induced by burst stimulation and the relationship between Vm and Ca2+ oscillations in VF were analyzed with cross-correlation analysis. The maximum correlation occurred at 12 ms delay between Vm and Cai, suggesting Vm still triggers Ca2+ release in VF as in normal excitation-contraction coupling. In addition, inverse correlation was found -20 ms between Vm and Cai, suggesting the amplitude of Cai can modulate action potential recovery in VF. In conclusion, Cai can influence action potential durations, which may promote wave breaks in VF.