Aim To document the safety and feasibility of intramuscular gene transfer by use of naked plasmid DNA encoding vascular endothelial growth factor and to observe potential therapeutic benefits in patients with critical limb ischemia. Methods Gene transfer was performed in 4 limbs of 3 patients with rest pain and nonhealing ischemic ulcers due to peripheral arterial disease. Four milligrams of naked plasmid DNA encoding the 165-amino-acid isoform of human vascular endothelial growth factor (pUC-CAGGS/h VEGF165) was injected directly into the muscles of the ischemic limb at 4 arbitrarily selected sites, two weeks apart. After 1～3 months after gene transfer, contrast angiography was repeated to document newly visible collateral blood vessels. Clinical status was recorded per day after gene transfer. Results Ischemic ulcers healed or markedly improved in 4 of 4 limbs, rest pain was relieved in all patients. Angiography confirmed newly visible collateral blood vessels in 4 of 4 limbs. Complications were limited to transient lower-extremity edema in all patients, consistent with VEGF enhancement of vascular permeability. Conclusions This findings may indicate that intramuscular injection of plasmid DNA expressing VEGF165 is sufficient to induce therapeutic angiogenesis in patients with critical limb ischemia. It may be a safe and effective strategy for patients with critical leg ischemia.