Antiviral cytotoxic T-cells are critical for control of lymphocytic choriomeningitis virus (LCMV) infection in mice. In H-2b mice, the antiviral response is directed against three Db-restricted epitopes in the viral nucleoprotein (NP396-404) and glycoprotein (GP276-286 and GP33-41). Our present data revealed a clear hierarchy among these three epitopes, in which NP396-404 is immunodominant, followed by GP33-41 and GP276-286, respectively. In order to identify additional CTL epitopes in the LCMV nucleoprotein and glycoprotein, we used the motifs for Db2- and Kb-binding peptides, combined with MHC class I-binding assays. Out of 23 Db motif-fitting peptides, we identified 4 Db binders, one of which (GP92-101) turned out to be a new CTL epitope. Among 28 Kb motif-fitting peptides, 12 bound Kb, and one of these (NP205-212) was a CTL epitope. Both newly identified CTL peptides were recognized by LCMV-immune splenocytes after secondary in vitro stimulation. Both peptides bound their MHC class I molecules with intermediate affinity (470 and 170 nM for GP92-101 and NP205-212, respectively). Responses against these peptides were weaker than the responses against the three major epitopes. None of the high affinity binders were new epitopes, suggesting that high affinity binders are either immunodominant epitopes or no epitopes at all. Thus, analysis of 51 Kb and Db motif-fitting peptides yielded 2 new, subdominant epitopes. Immunization of C57BL/6 mice with these peptides, or vaccinia virus recombinants expressing these epitopes as minigenes, protected against chronic LCMV infection, demonstrating that immunization with subdominant epitopes can confer protection against chronic viral infection.