Objectives: The therapeutic utility of hypoxia-inducible factor-1 (HIF-1) transcriptional regulatory system for ischemic hindlimb has been demonstrated. It is not yet known whether this transcriptional regulatory system can be used as a therapeutic strategy to enhance collateral vessel formation in myocardial tissues, where acute hypoxia occurs due to inadequate perfusion. We aimed to test the hypothesis that exogenous administration of HIF-1α/VP16 could enhance collateral vessel formation in a rat acute myocardial infarction model. Methods: Sprague–Dawley rats received ligation of the proximal left anterior descending coronary artery to induce acute myocardial infarction. Immediately after the ligation, 50 μg total plasmid DNA (control, plasmid encoding human vascular endothelial growth factor (pVEGF₁₆₅), or pHIF-1α/VP16) was injected into the infarct area at three locations. Results: Reverse transcription–polymerase chain reaction (RT–PCR) showed the presence of HIF-1α and VEGF mRNA in the myocardium, but not in other organs at days 3 and 7. The infarct size significantly decreased from 37±4% (control) to 24±2% in the VEGF-treated group and 23±2% in the HIF-1α/VP16 treated group (P<0.05). Capillary density also significantly increased from 550±75/mm² (control) to 850±75/mm² in the VEGF group and 850±50/mm² in the HIF-1α/VP16-treated group (P<0.01). Combined therapy with HIF-1α/VP16 and VEGF resulted in higher capillary density (1230±50/mm²) than treatment with either therapy alone. Regional myocardial blood flow was also higher in the treated groups than in the control. Plasma levels of VEGF were also significantly higher in the HIF-1α/VP16 and VEGF-treated group than in the control group. Conclusions: The HIF-1α/VP16 hybrid transcription factor is able to reduce infarct size and enhance neovascularization in an acute ischemic myocardium. The potency of VEGF and HIF-1α/VP16 hybrid as therapeutic angiogenic factors in acute hypoxic myocardium is similar.