Efficient pulmonary vascular gene transfer in neonates would aid in understanding the pathophysiology of, and ultimately allow the development of specific therapies for, pulmonary vascular diseases. The purpose of this study was to optimize efficiency, and evaluate the duration, of catheter-based adenoviral vector-mediated pulmonary artery gene transfer in newborn pigs. An adenovirus vector encoding LacZ was infused via percutaneously placed catheters that were advanced to segmental pulmonary arteries under fluoroscopic guidance. Optimal viral dose and duration of expression were determined by histochemical evaluation of gene transfer efficiency 72 hr, 2 weeks, and 1 month after gene delivery. The effect of protamine on the efficiency of gene transfer was studied by assaying transgene protein in lung at 72 hr. The optimal viral dose was 2 x 10(10) plaque-forming units (PFU). Seventy-two hours after infusion, expression predominated in medium-sized artery endothelial cells, 40% of which expressed beta-galactosidase. At 2 weeks, the distribution of expression had changed such that the majority of transduced cells were seen not in arteries but in gas exchange units of lung. No histochemical evidence of transgene expression was seen 1 month after virus infusion. The addition of protamine to virus infusate resulted in a fivefold increase in transgene protein product in lung tissue assayed 72 hr after gene transfer. Adenoviral vector-mediated gene transfer in neonatal swine results in high-efficiency transduction of arterial endothelial cells. However, the time course of gene transfer is not significantly prolonged compared with the adult. The addition of protamine results in a significant improvement in transduction efficiency, permitting lower doses of virus.