We set out to test the hypothesis that IL-22, a cytokine crucial for epithelial cell homeostasis and recovery from tissue injury, would be protective during influenza virus infection. Recent studies have identified phenotypically and functionally unique intestinal NK cells capable of producing the cytokine IL-22. Unlike gut NK cells that produce IL-22, the surface phenotypes of the lung NK cells were similar to spleen NK cells and characteristically mature. With mitogen stimulation, both single and double IL-22 and IFN-gamma lung producing NK cells were detected. However, only the IL-22+IFN-gamma- lung NK subset was observed after stimulation with IL-23. IL-23 receptor (IL-23R) blocking dramatically inhibited IL-22 production, but not IFN-gamma. Further, we found NK1.1+ or CD27- lung NK cells were the primary sources of IL-22. After influenza virus infection, lung NK cells were quickly activated to produce both IFN-gamma and IL-22, and had increased cytotoxic potential. The level of IL-22 in the lung tissue declined shortly after infection, gradually returning to baseline after virus clearance, although the IL-22 gene expression was maintained. Further, depletion of NK cells with or without influenza infection reduced the protein level of IL-22 in the lung. Anti-IL-22 neutralization in vivo did not dramatically affect weight loss and survival after virus clearance. Unexpectedly, anti-IL-22 treated mice had reduced virus titers. Our data suggest that during primary respiratory viral infection, IL-22 seems to play marginal role for protection, indicating a differential requirement of this cytokine for bacterial and viral infections.