BACKGROUND-: Hypoxia-inducible factor-1α (HIF-1α) is a transcriptional regulatory factor that orchestrates cellular responses to hypoxia. It increases collateral vessel growth and blood flow in models of hind-limb ischemia. This study tested whether intramuscular administration of Ad2/HIF-1α/VP16, an engineered recombinant type 2 adenovirus vector encoding constitutively active HIF-1α, improves walking time in patients with peripheral artery disease and intermittent claudication. METHODS AND RESULTS-: Two hundred eighty-nine patients with claudication were randomized in a double-blind manner to 1 of 3 doses of Ad2/HIF-1α/VP16 (2×10, 2×10, or 2×10 viral particles) or placebo, administered by 20 intramuscular injections to each leg. Graded treadmill tests were performed at baseline and then 3, 6, and 12 months after treatment. The primary end point was the change in peak walking time from baseline to 6 months. The secondary end point was change in claudication onset time, and tertiary end points included changes in ankle-brachial index and quality-of-life assessments. Median peak walking time increased by 0.82 minutes (interquartile range,-0.05-1.93 minutes) in the placebo group and by 0.82 minutes (interquartile range,-0.07-2.12 minutes), 0.28 minutes (interquartile range,-0.37-1.70 minutes), and 0.78 minutes (interquartile range,-0.02-2.10 minutes) in the HIF-1α 2×10, 2×10, and 2×10 viral particle groups, respectively (P=NS between placebo and each HIF-1α treatment group). There were no significant differences in claudication onset time, ankle-brachial index, or quality-of-life measurements between the placebo and each HIF-1α group. CONCLUSIONS-: Gene therapy with intramuscular administration of Ad2/HIF-1α/VP16 is not an effective treatment for patients with intermittent claudication.