We recently could demonstrate, that local eNOS S1177D (D, constitutive active) transfection is feasible in a model of acute myocardial ischemia. In the present study we used selective pressure regulated retroinfusion as an approach to target eNOS cDNA into chronic ischemic myocardium. We investigated the influence of eNOS D on myocardial function and perfusion. Pigs were subjected to percutaneus implantation of a reduction stent into the LAD, to induce a high-grade stenosis (total occlusion at day 28). After 28d pigs were treated retrogradly with either eNOS cDNA +- oral application of a NO-Inhibitor or saline solution. Assessment of regional myocardial blood flow global myocardial function, regional myocardial function, collateral and capillary score were conducted and immunohistology was performed. furthermore we investigated the eNOS expression and the amount of proliferated cells. eNOS D retroinfusion lead to an increased capillary and collateral growth, enhanced regional myocardial perfusion and function. This effects were abolished through co-application of L-NAME. eNOS expression was enhanced in the ischemic tissue compared to the non ischemic, after transfection. Through the eNOS transfection proliferation of endothelial cells and smooth muscle cells was enhanced. Transfection eNOS D enhances neovascularization at both, the microcirculatory and macrocirculatory level and improves blood perfusion, regional function of hibernating myocardium.