Background. Experiments were designed to study the efficiency, distribution, and toxicity of transbronchial adenoviral-mediated transfer of endothelial constitutive nitric oxide synthase (ecNOS) gene to transplanted lungs. Methods. Syngeneic orthotopic single-lung transplantation in the rat was performed after airway administration (300 μL, 1 x 109 pfu/mL) of either the ecNOS gene or the marker gene β-Gal (control group) to donor lungs (n = 4 each). After 4 days, transgene expression, inflammation, and the presence of apoptosis in the transplanted lungs were assessed by molecular, immunohistochemical, and histologic techniques. Results. Gene transfer was confirmed by a positive polymerase chain reaction signal for the recombinant ecNOS gene, and recombinant messenger RNA by reverse transcription polymerase chain reaction. Positive immunohistochemical staining for ecNOS was present in more than 75% of pneumocytes only in ecNOS transduced lungs. Calcium- dependent nitric oxide synthase activity was increased in ecNOS- compared with βGal-transduced lungs (2,139 ± 756 versus 47 ± 28 pmol · mg protein-1 · h-1; p < 0.05). Minimal to mild inflammation was observed in all transplanted lungs; fewer than 0.5% of cells in both groups were apoptotic. Conclusions. Transbronchial transfer of ecNOS gene to the transplanted lung results in transduction of pneumocytes with expression of a functionally active transgene product.