We hypothesized that adenovirus-mediated inducible nitric oxide synthase (iNOS) gene transduction of the lung would result in time-dependent iNOS over-expression and attenuate the vascular constrictor responses to a thromboxane mimetic, U-46619. Rats were treated via the trachea with surfactant alone (sham), surfactant containing an adenoviral construct with a cytomegalovirus promoter regulated human iNOS gene (Adeno-iNOS) or an adenoviral construct without a gene insert (Adeno-Control). Adeno-iNOS transduced rats demonstrated human iNOS (hiNOS) mRNA, and increased iNOS protein levels only in the lungs. Immunohistochemistry of lungs from Adeno-iNOS treated animals demonstrated transgene expression in alveolar wall cells. In the lungs from Adeno-iNOS transduced rats the expression of iNOS protein and exhaled NO (exNO) concentrations were increased on days 1-4 and 7, but returned to baseline values by day 14. The administration of the selective iNOS inhibitor L-N⁶-(1-iminoethyl)lysine dihydrochloride (L-NIL) decreased exNO concentrations to levels found in Adeno-Control transduced lungs. In a second group of rats the segmental vasoconstrictor responses to U-46619 were determined in isolated, perfused lungs 3 days following transduction. Lungs from rats transduced with Adeno-iNOS had reduced total, arterial and venous vasoconstrictor responses to U-46619 compared to sham, Adeno-Control, and control groups. In a third set of experiments the response to 400 nM U-46619 in the presence of 10 µM L-NIL was not different in the isolated lungs from Adeno-Control and Adeno-iNOS transduced rats. We conclude that adenovirus-mediated iNOS gene transduction of the lung results in time-dependent iNOS over-expression, which attenuates the vascular constrictor responses to the thromboxane mimetic, U-46619.