Skin ischemic necrosis due to vasospasm and/or insufficient vascularity is the most common complication occurring in the distal portion of the skin flap in reconstructive surgery. This project was designed to test our hypothesis that preoperative subdermal injection of adenoviral vectors encoding genes for endothelial growth factor-165 (Ad.VEGF₁₆₅) or endothelial nitric oxide synthase (Ad.eNOS) is effective in augmentation of skin viability in skin flap surgery and the mechanism of Ad.VEGF165 gene therapy involves an increase in synthesis/release of the angiogenic and vasodilator factor nitric oxide (NO). The 4 x 10 cm rat dorsal skin flap model was used in all studies. Phosphate buffered saline (PBS, 0.5 ml), or PBS containing Ad.VEGF_165M, Ad.eNOS, or adenovirus (Ad.Null) was injected subdermally in the distal half of the mapped rat dorsal skin flap 7 days preoperatively. Skin flap viability was assessed 7 days postoperatively. It was observed that local subdermal VEGF_165M gene therapy increased skin flap viability at the dose range of 2 x 10⁷ - 2 x 10¹⁰ pfu, compared with the PBS or PBS and Ad.Null injected control (p < 0.05). Subdermal Ad.VEGF_165M and Ad.eNOS gene therapy were equally effective in increasing skin flap viability at the dose tested (5 x 10⁸ pfu). Subdermal Ad.VEGF_165M therapy was associated with an upregulation of eNOS protein expression, calcium-dependent NOS (cNOS) activity, and synthesis/release of NO and an increase in capillary density and skin blood flow in the distal portion of the skin flap. Furthermore, intramuscular injection of the NOS inhibitor N - nitro - L - arginine (15 mg/kg) but not the cyclooxygenase inhibitor indomethacin (5 mg/kg) at 45 min preoperatively completely abolished the increase in skin flap blood flow and viability induced by local subdermal injection of Ad.VEGF_165M 7 days preoperatively. In conclusion, we have demonstrated for the first time that: (1) local subdermal injection of Ad.VEGF_165M or Ad.eNOS 7 days preoperatively are equally effective in augmenting skin viability in the rat dorsal skin flap compared with the control; (2) the mechanism of subdermal Ad.VEGF_165M gene therapy in augmenting skin flap viability involves an increase in synthesis/release of NO, downstream to upregulation of eNOS protein expression and cNOS activity; and (3) the vasodilating effect of NO may predominantly mediate the subdermal Ad.VEGF gene therapy in augmenting skin flap blood flow and viability.