The viral antigen specificity of primary cytotoxic T cell responses (CTL) of H-2b, H-2k, H-2q, H-2s, H-2f and some H-2-recombinant mice against lymphocytic choriomeningitis virus (LCMV-WE isolate) as well as the specificity of some CTL clones and T cell lines was defined on target cells infected with vaccinia-recombinant virus expressing nucleoprotein (Np) or glycoprotein (Gp). Np was recognized together with H-2q (Dq), H-2d (DLd), H-2s and H-2b (Db). Gp specificity was restricted to H-2f and H-2b (Kb and Db); H-2k-restricted CTL anti-LCMV responses were neither Gp nor Np specific. The anti-viral protective immunity induced by vaccinia-Gp or vaccinia-Np recombinants was evaluated in mice. In vivo protection was T cell mediated by class I restricted Ly-2+ T cells; it correlated well with the CTL specificity defined in vitro. Some of the CTL-nonresponder H-2 allele plus Np or H-2 plus Gp combinations were, however, protected to variable and low degrees by vaccinia-recombinant viruses, indicating that anti-viral protection is a more sensitive readout for CTL activity than the in vitro assay. For example, B10.D2 H-2d mice generated measurable CTL responses only to Np; after immunization with a vaccinia-Np recombinant, LCMV titers were 10(4) times lower in spleens than in vaccinia-primed controls. Although vaccinia-Gp-immunized BALB/c mice revealed no CTL activity in vitro, they nevertheless had 10(2) times lower LCMV titers in spleens than controls. Anti-viral protection, particularly in low-responder combinations, was usually short-lived and diminished after 3 weeks. In a high-responder situation, protection was of a longer duration (greater than 8 weeks). Vaccination with vaccinia-Np or Gp recombinants protected mice against lethal T cell-mediated lymphocytic choriomeningitis induced by LCMV or prevented the local footpad swelling reaction; these in vivo effects were H-2 dependent and followed the identical roles established for CTL recognition in vitro.