Abstract Coronary artery disease (CAD), a leading cause of mortality, is a chronic disease in which blood flow to the myocardium is obstructed, leading to ischemia. Although coronary artery stenting and surgical bypass are successful with localized coronary lesions, patients with diffuse CAD have only pharmacologic options. In a mouse ischemic hind-limb model, AdVEGF-All6A+, an Ad5 vector expressing the cDNA/genomic hybrid of the vascular endothelial growth factor (VEGF) gene (expressing isoforms, 121, 165, and 189) mediated recovery of blood flow at a dose of two logs less than that required with a single isoform. The objective of the current study was to ascertain the safety profile of good manufacturing practice (GMP)-grade AdVEGF-All6A+ in the adult rat ischemic heart model in support of a clinical study to treat humans with diffuse CAD. AdVEGF-All6A+ (10(5), 10(6), or 10(7) particle units), a control vector (AdNull, 10(7) particle units) with no translatable expression cassette and a vehicle sham control (phosphate buffered saline [PBS]) were administered separately to the left ventricle of rats immediately following acute coronary artery ligation to initiate myocardial infarction (MI), designed to evoke an extreme ischemic myocardium in cohorts (n=5 males; n=5 females), with sacrifice at 5, 14, or 30 days. Six cohorts received no ligation but were administered AdVEGF-All6A+ vector or PBS with sacrifice at 30 or 365 days. Although there were surgical-related abnormalities among the groups, blinded evaluation of gross and histopathology, complete blood count, and serum chemistry found no significant differences between control- and vector-treated groups and no adverse effects could be attributed to AdVEGF-All6A+. No changes in serum troponin-I levels persisted beyond those associated with the MI. Gross pathology and histopathology of all major organs showed no AdVEGF-All6A+-related changes. Overall, this safety profile suggests that AdVEGF-All6A+ or AdNull administration to the myocardium meets the criteria to proceed to clinical trial.