We reported previously that adoptive immunization with an influenza A virus NS1-specific H-2Ld-restricted, cross-reactive, CTL clone A-11 established by stimulation with A/PR/8/34 virus (H1N1) reduced lung virus titers in mice challenged with virus in vivo (Virology 178:174-179, 1990). Using a set of recombinant vaccinia virus constructs containing truncated portions of the NS gene we have localized this cross-protective CTL epitope to the N-terminal region of the NS1 protein. This region of NS1 is active in inducing CD8+ CTL in vivo because virus-stimulated BALB/c immune spleen cells in bulk cultures also recognized the N-terminal region of the NS1 protein.