Highly pathogenic influenza A viruses including avian H5N1 viruses and the 1918 pandemic virus cause severe respiratory disease in humans and animals. Virus infection is followed by intense pulmonary congestion due to an extensive influx of macrophages and neutrophils which can release large quantities of reactive oxygen species potentially contributing to the pathogenesis of lung disease. Here the role of nitric oxide (NO), a potent signaling molecule in inflammation was evaluated following highly pathogenic influenza virus challenge in mice. We observed higher levels of NO in mice infected with H5N1 and 1918 viruses compared to a seasonal H1N1 virus. Mice deficient in inducible nitric oxide synthase (NOS2(-/-)) exhibited reduced morbidity, mortality and diminished cytokine production in lung tissue following H5N1 and 1918-virus challenge compared with wild-type control mice. Furthermore, systemic treatment of mice with the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) delayed weight loss and death among 1918 virus infected mice compared to untreated control animals. This study demonstrates that NO contributes to the pathogenic outcome of H5N1 and 1918 viral infections in the mouse model.