Therapeutic angiogenesis involves the introduction of exogenous growth factor proteins and genes into ischemic tissues to augment endogenous factors and promote new vessel growth. Positive results from studies in animal models of peripheral arterial disease (PAD) and coronary artery disease over the past decade have supported the implementation of clinical trials testing vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) proteins and genes. Although several clinical trials reported positive results, others have been disappointing and results of a recent Phase II trial of VEGF delivered by adenovirus (the RAVE trial) were negative. It has been suggested that the duration of gene expression following delivery by adenovirus may be insufficient to produce stable vessels. Here we present direct evidence in support of this using the rabbit ischemic hindlimb model injected with adenovirus encoding VEGF165. Immunohistology indicated an activation of endothelial cell cycling and proliferation 2-3 days after VEGF delivery that coincided closely with transient VEGF expression. Ki-67-positive endothelial nuclei were evident at high levels in capillaries and large vessels in muscles from treated animals. Angiography indicated increased density of both large and small vessels in Ad-VEGF-treated muscle at 1 week, but no significant differences thereafter. The early burst of endothelial proliferation was accompanied by increased nuclear fragmentation and condensation in VEGF-treated muscles, suggesting coincident apoptosis. No further endothelial cell proliferation took place after 1 week although there was still evidence of apoptosis. The results suggest that angiogenesis is confined to the short period of VEGF expression produced by adenovirus and early gains in collateralization rapidly regress to control levels when VEGF production ceases.