Hypoxia-inducible factor (HIF)-1alpha and-2alpha have diverse actions on the myocardium, but the importance of direct effects on cardiac myocytes is unclear. To define their regional accumulation and association with cardiomyocyte cell cycle change after myocardial infarction (MI), a rat MI model was established by occluding the coronary arteries. To further prove a causative relationship between HIF and cell cycle regulation, cultured cardiomyocytes were transfected with adenoviral vectors carrying HIF-1alpha and HIF-2alpha. Two weeks after MI, both HIF-1alpha and HIF-2alpha mRNA were moderately increased in the infarcted left ventricle and noninfarcted left ventricle; HIF-2alpha amplification was also detected in areas of the interventricular septum and the right ventricle. In concordance with the changes in mRNA levels, immunohistochemistry signals of HIF-1alpha and HIF-2alpha were characterized by different regional distributions. In the myocardium adjacent to the infarcted tissue, a significant correlation between HIF-1alpha or HIF-2alpha and Ki-67 labeling index was observed (P < 0.001). Immunohistochemical double staining showed that HIF positive cardiomyocytes underwent DNA synthesis. Cardiomyocytes treated with HIF-1alpha or -2alpha expressed Ki-67, phosphohistone H3, and bromodeoxyuridine effectively in vitro. In conclusion, HIF-1alpha and HIF-2alpha had a distinct spatial expression pattern in a rat model of ischemic heart disease. Both HIF subunits might be potent stimuli for cardiomyocytes to re-enter the cell cycle and initiate DNA synthesis.