Introduction: Acute myocardial ischemia, which opens KATP channel, is associated with shortened action potential duration (APD) but prolonged QT interval. This discrepancy has not been adequately explained. We hypothesize that the duration of intracellular calcium (Cai) transient (DCaT) may play a role in determining QT interval. Methods and Results: We performed simultaneous optical mapping of voltage and Cai in 15 isolated rabbit hearts during a KATP channel opener (pinacidil) infusion. Anterior epicardial mapping (n = 7) showed no difference of APD90, QT interval, and the DCaT90 at baseline. When perfused with 80 μM pinacidil, the APD90, the QT interval, and the DCaT90 were 105 ± 10 msec, 199 ± 14 msec, and 189 ± 13 msec, respectively, during right ventricular (RV) pacing (P < 0.05). Posterior epicardial mapping (n = 4) showed that the APD90 was significantly (P < 0.05) shorter than QT interval and DCaT90 during pinacidil infusion. The results of the transmural mapping studies (n = 4) showed that the QT interval during RV pacing was not different than the DCaT90 in the epicardium, midmyocardium, and endocardium, but was significantly (P < 0.01) longer than the APD90 in epicardium, midmyocardium, and endocardium, respectively. There was a good correlation between the DCaT90 and QT interval at baseline (r = 0.92, P < 0.0001) and during pinacidil infusion (r = 0.74, P < 0.0001). Conclusion: We conclude that KATP channel opening shortened APD but not the QT interval. Because Cai did not return to diastolic level at the end of action potential, it may have created a heterogeneous membrane potential distribution that determined the QT interval.