Background Vascular endothelial growth factor (VEGF) currently is being evaluated in clinical angiogenesis trials involving patients with peripheral arterial disease. We hypothesized that delivery of VEGF to the skeletal muscle of the lower extremity using an adenoviral vector (Ad GV VEGF 121.10 ) would improve peripheral endothelial function. Accordingly, we investigated lower-extremity endothelial function in patients enrolled in a Phase I adenovirus-mediated gene delivery trial of VEGF 121.10 . Methods and Results Blood flow to the index extremity was measured by thermodilution at baseline and 30 days after administration of Ad GV VEGF 121.10 , in response to the infusion of endothelium-dependent and -independent agonists (acetylcholine and nitroglycerin, respectively) into the ipsilateral femoral artery. There was no difference in basal flow before or after treatment with Ad GV VEGF 121.10 . In response to acetylcholine (150 μg/min and 300 μg/min), there was a 0.9-fold (0.33±0.03 to 0.32±0.03 L/min) and 1.2-fold (0.33±0.03 to 0.490±0.02 L/min) change in flow before Ad GV VEGF 121.10 treatment. After Ad GV VEGF 121.10 treatment, flow increased 2.4-fold (0.310±0.04 to 0.730±0.10 L/min) and 2.3-fold (0.31±0.04 to 0.7±0.08 L/min), respectively ( P <0.05 before Ad GV VEGF 121.10 treatment versus after Ad GV VEGF 121.10 for both doses). Infusion of nitroglycerin resulted in a 1.8-fold increase in flow before Ad GV VEGF 121.10 (0.33±0.03 to 0.58±0.06 L/min) compared with a 2.4-fold increase (0.31±0.04 to 0.73±0.09 L/min) after Ad GV VEGF 121.10 ( P =NS before Ad GV VEGF 121.10 versus after Ad GV VEGF 121.10 ). Lower-extremity flow reserve increased in all patients in response to at least 1 dose of acetylcholine. Peak walking times increased concomitant with improvement in endothelial function. Conclusions Adenoviral gene transfer of VEGF 121.10 appears to modulate endothelial function and lower-extremity flow reserve in patients with peripheral arterial disease.