We previously reported that the epitope recognized by an influenza A virus H1, H2, and H3-crossreactive, H-2 Ld-restricted CD8+ cytotoxic T lymphocyte (CTL) is located between amino acids 1 and 40 on the nonstructural protein NS1. In the present experiments, we examined whether immunization with recombinant vaccinia virus which contained genes coding for amino acids 1-40 of NS1 (Vac-10) protected mice from lethal challenge with influenza A virus. Mice immunized with this recombinant virus developed influenza A virus-specific cytotoxic activity but not neutralizing antibodies. Challenge with a lethal dose of influenza A virus demonstrated that the first deaths were delayed by 2 days, and the mortality rate was significantly reduced (p < 0.05) in Vac-10-immunized mice compared with mice immunized with control vaccinia virus. These results suggest that immunization with a single subtype-crossreactive CTL epitope on NS1 can induce protective immunity against lethal influenza A virus infection.