We assessed the role of ·NO in recombinant adenovirus-mediated gene transfer both in vitro and in vivo. NIH3T3 fibroblasts, stably transfected with the human inducible nitric oxide synthase, but lacking tetrahydrobiopterin (NIH3T3/iNOS [inducibile nitric oxide synthase]), were infected with replication-deficient adenovirus (E1-deleted), containing either the luciferase or the Lac Z reporter genes (AdCMV-Luc and AdCMV-Lac Z; 1-10 plaque forming units [pfu]/cell). Incubation of infected cells with sepiapterin (50 μM), a precursor of tetrahydrobiopterin, progressively increased nitrate/nitrite levels in the medium and decreased both luciferase and β-galactosidase protein expression to approximately 60% of their corresponding control values, 24 h later. NIH3T3/iNOS cells had normal ATP (adenosine 5′-triphosphate) levels and did not release LDH(lactic dehydrogenase) into the medium. Pretreatment of these cells with NG-monomethyl-L-arginine (L-NMMA; 1 mM), an inhibitor of iNOS, prevented the sepiapterin-mediated induction of ·NO and restored gene transfer to baseline values. Incubation of NIH3T3/iNOS with 8-bromo-cGMP (400 μM) in the absence of sepiapterin, or exposure of AdCMV-Luc to large concentrations of ·NO, did not alter the efficacy of gene transfer. ·NO produced by NIH3T3/iNOS cells also suppressed β-galactosidase expression in NIH3T3 cocultured cells stably transfected with β-galactosidase gene, suggesting ·NO inhibited gene expression at either the transriptional or posttranscriptional levels. To investigate the effects of inhaled ·NO on gene transfer in vivo, CD1 mice received an intratracheal instillation of AdCMV-Luc (4 × 109 pfu in 80 μl of saline) and exposed to ·NO (25 ppm in room air) for 72 h. At that time, no significant degree of lung inflammation was detected by histological examination. However, lung luciferase activity decreased by 53% as compared with air breathing controls (P < 0.05; n ≥ 8). We concluded that overproduction of ·NO decreases the efficiency of adenovirus-mediated gene transfer in lung cells in the absence of cytotoxicity or inflammation.