Objective: Most currently available percutaneous delivery methods for arterial gene therapy are limited by the need for a long incubation period, which may lead to unacceptable tissue ischemia, especially in the coronary vasculature. Conversely, shorter incubation times may result in inefficient gene transfer, especially in atheromatous arteries. A new local delivery autoperfusion multichamber catheter is now available which permits local delivery in the coronary arterial system without inducing myocardial ischemia. The present study aimed at evaluating the performance of this catheter for achieving arterial gene transfer using replication-defective adenoviral vectors in normal and atheromatous arteries. Methods: A replication-defective adenoviral vector carrying a nuclear-targeted β-galactosidase reporter gene (Ad-RSVβgal, 5.10[9] plaque-forming units [pfu]) was delivered to the iliac arteries of normal (n = 7) and atheromatous (1% cholesterol diet + arterial abrasion) (n = 6) rabbits, via a multichamber autoperfusion balloon catheter (DispatchTM, SciMed). Duration of gene delivery was 60 min. Results: Three days later, marked expression of the reporter gene was detected by histochemistry in the endothelium at the delivery site (percentage of transfected cells: 16 ± 8% /artery (range 11–25%). There was a low transduction rate in medial smooth muscle cells 0.7 ± 0.4% /artery (range 0.3–1.1%). In atheromatous arteries, transduction was consistently achieved in the superficial layers of the neointima but was lower (1.1 ± 0.5% /artery, range 0.3–1.7%). Transgene expression was detected by histochemistry in the liver of 3/13 animals, suggesting that there is a substantial risk of systemic dissemination of the viral vectors. Conclusion: Efficient arterial gene delivery to endothelial and superficial smooth muscle cells is feasible using local delivery of adenoviral vectors via the DispatchTM autoperfusion catheter, in both normal and atheromatous arteries. This perfusion catheter may be a useful tool for coronary artery gene transfer.