Background: The Histidine-Tryptophan-Ketoglutarate (HTK) solution has been shown to provide the excellent myocardial protection as a cardioplegia. The HTK solution has relatively low potassium as an arresting agent of myocardium, and low sodium content, and high. concentration of histidine biological buffer which confer a buffering capacity superior to that of blood.. Since HTK solution has an excellent myocardial protective ability, it is reported to protect myocardium from ischemia for a considerable time (120 minutes) with the single infusion of HTK solution as a cardioplegia. The purpose of this study is to evaluate the cardioprotective effect of HTK solution on myocardium when the ischemia is. exceeding 120 minutes at two different temperature (10 to 12$^{\circ}C$, 22 to 24$^{\circ}C$) using the Langendorff apparatus, Material and Method: Hearts from Sprague-Dawley rat, weighing 300 to 340 g, were perfused with Krebs-Henseleit solution at a perfusion pressure of 100 cm $H_2O$. After the stabilization, the heart rate, left ventricular developed pressure (LVDP), and coronary flow were measured. Single dose of HTK solution was infused into the ascending aorta of isolated rat heart and hearts were preserved at four different conditions. In group 1 (n=10), hearts were preserved at deep hypothermia (10∼12$^{\circ}C$) for 2 hours, in group 2 (n=10), hearts were preserved at moderate hypothermia (22∼24$^{\circ}C$) for 2 hours, in group 3 (n=10), hearts were preserved at deep hypothermia for 3 hours, and in group 4 (n=10), hearts were preserved at moderate hypothermia for 3 hours. After the completion of the preservation, the heart rate, left ventricular developed pressure, and coronary flow were measured at 15 minutes, 30 minutes, and 45 minutes after the initiation of reperfusion to assess the cardiac function. Biopsies were also done and mitochondrial scores were counted in two cases of each group for ultrastructural assessment. Result: The present study showed that the change of heart rate was not different between group 1 and group 2, and group 1 and group 3. The heart rate was significantly decreased at 15 minutes in group 4 compared to that of group 1 (p<0.05 by ANCOVA). The heart rate was recovered at 30 minutes and 45 minutes in group 4 with no significant difference compared to that of group 1. The decrease of LVDP was significant at 15 minutes, 30 minutes and 45 minutes in group 4 compared to that of group 1 (p < 0.001 by ANCOVA). Coronary flow was significantly decreased at 15 minutes, 30 minutes, and 45 minutes in group 4 compared to that of group 1 (p < 0.001 by ANCOVA). In ultrastructural assessment, the mean myocardial mitochondrial scores in group 1, group 2, group 3, and group 4 were 1.02$\pm$0.29, 1.52$\pm$0.26, 1.56$\pm$0.45, 2.22$\pm$0.44 respectively. Conclusion: The HTK solution provided excellent myocardial protection regardless of myocardial temperature for 2 hours. But, when ischemic time exceeded 2 hours, the myocardial hemodynamic function and ultrastructural changes were significantly deteriorated at moderate hypotherma (22∼ 24$^{\circ}C$). This indicates that it is recommended to decrease myocardial temperature when myocardial ischemic time exceeds 2 hours with single infusion of HTK solution as a cardioplegia.