Objective. Angiogenic therapy with individual growth factors or 'master switch' genes is being evaluated for treatment of advanced coronary artery disease. In this study we investigated the efficacy and mechanism of PR39, a gene capable of activating vascular endothelial growth factor (VEGF)- and fibroblast growth factor (FGF)-2-dependent pathways. PR39 enhances HIF-1-dependent gene expression by selectively inhibiting proteasome degradation of this transcription factor. In addition, PR39 also stimulates expression of the fibroblast growth factors receptors FGFR-1 and syndecan-4. Methods and Results. In a pig model of chronic myocardial ischemia, we used angiography, MRI and microsphere regional blood flow (RBF), to evaluate the efficacy of intramyocardial Ad-PR39 injections. Ad-PR39 improved collateral scores, regional perfusion and regional function in a dose-dependent manner. Local VEGF, VEGFR-1, VEGFR-2, syndecan and FGFR-1 levels were 16-75% upregulated following Ad-PR39 injections as assessed by real time PCR suggesting upregulation of VEGF and FGF pathways. Conclusion. PR39 is an angiogenic peptide that improves perfusion and function of ischemic myocardium at least in part through collateral formation. The dual mechanism, i.e. stimulation HIF-1 and FGF receptor expression, likely accounts for the functional benefits of PR39.