α1-Antitrypsin (α1AT) is a serine proteinase inhibitor that protects the lung from degradation by neutrophil proteases. In α1AT deficiency, an autosomal recessive disorder resulting from mutations in the α1AT (approved symbol SERPINA1) gene, serum α1AT levels of < 570 μg/ml are associated with development of emphysema. Adeno-associated virus (AAV) serotype 2 (AAV2) vectors expressing α1AT administered intramuscularly or intravenously mediate sustained serum levels of α1AT in experimental animals. Since the lung is only 2% of the body weight, AAV vector delivery to the muscle or liver is inefficient, as most of the α1AT does not reach the lung. The present study evaluates AAV2- and AAV5-mediated delivery of human α1AT (hα1AT) to C57BL/6 mice using the intrapleural space as a platform for local production of α1AT. Intrapleural administration of either an AAV5-hα1AT or an AAV2-hα1AT vector achieves higher lung and serum levels of α1AT than intramuscular delivery. AAV5-mediated serum and lung α1AT levels were 10-fold higher than those achieved by AAV2 delivery via either route. The diaphragm, lung, and heart are the major sites of transgene expression following intrapleural administration of an AAV5 reporter vector. At 40 weeks postadministration, intrapleural administration of the AAV5-hα1AT vector mediated serum α1AT levels of 900 ± 50 μg/ml, 1.6-fold higher than the accepted therapeutic level of 570 μg/ml. In the context that the pleura is a safe site for administration, intrapleural administration using AAV5 vectors may represent an attractive gene therapy strategy for α1AT deficiency in humans.