Inadequate antiprotease activity in the lungs due to alpha-1-antitrypsin (A1AT) deficiency is a factor of early-onset emphysema. We propose a new approach to gene therapy that involves the intratracheal delivery of macrophages expressing human A1AT (hA1AT). Recombinant adeno-associated virus (rAAV) plasmids encoding the hA1AT gene were packaged into virions using 293 cells, and transgenic progeny virus was purified from the cells. The murine macrophage cell line J774A.1 was infected in vitro with the recombinant hA1AT rAAV virus. The hA1AT-producing macrophages were delivered intratracheally into mechanically ventilated C57BL/6J mice, a strain with low endogenous levels of A1AT. Transcription of hA1AT mRNA was detected in the transfected cells by RT-PCR, and protein expression was verified by immunohistochemistry. Levels of hA1AT in the cell culture medium and in the bronchoalveolar lavage (BAL) were assayed by ELISA. The concentration of hA1AT in J774A.1 cell-conditioned medium increased from undetectable levels prior to transfection, to 60 mg/l at 24 h post-transfection. At 1, 3 and 7 days after intratracheal delivery of transfected macrophages, hA1AT protein in BAL from C57BL/6J mice increased from undetectable levels to 2.5+/-0.9, 2.6+/-1.1 and 2.2+/-0.8 mg/l, respectively. These results suggest that airway delivery of macrophages overexpressing hA1AT may be an effective approach to enhance alveolar protection in A1AT deficiency.