Protection against influenza A virus infection in mice immunized with recombinant nucleoprotein (rNP) was studied. Nucleoprotein-immune mice were protected against a lethal challenge with A/Puerto Rico/8/34 (A/PR8) virus but showed considerable morbidity before recovery. Local boosting of the immune system with rNP by intranasal immunization improved the protection in NP-immune mice, and the decrease in morbidity after infection was reflected in accelerated clearance of virus from lungs. However, immune, boosted mice also rapidly cleared an antigenically unrelated influenza B virus from their lungs. Mice immunized with rNP precipitated with alhydrogel, that subsequently developed significant resistance to virus infection, failed to generate detectable levels of class I major histocompatibility complex (MHC)-restricted killer cells. Furthermore, B10.A(5R) mice that are non-responders for NP-specific class I killer cells could also be protected by immunization with rNP. In contrast, rNP-immunized mice developed strong proliferative T-cell responses to rNP. These data argue for an important role for helper T cells rather than virus-specific class I cytotoxic T cells in protection against influenza virus infection induced by rNP.