Mice expressing the H2b haplotype are resistant to infection with Theiler's murine encephalomyelitis virus (TMEV), which causes chronic demyelination in susceptible mice. The prominent cytolytic T-lymphocyte (CTL) response to the VP2 antigen encoded by TMEV led us to the identification of a class I-binding peptide derived from the VP2 antigen. Escherichia coli transformants overexpressing a series of 11 overlapping VP2 protein fragments were subjected to lysis and alkali digestion, and the resultant peptide pools were tested for their abilities to sensitize RMA-S targets for lysis by CTLs. The source of effector CD8+ T cells for the assays was either freshly harvested central nervous system-infiltrating lymphocytes (CNS-IL) or CNS-IL-derived VP2-specific CTL clones and lines. A 10-residue peptide at VP2 positions 121 to 130 (VP2(121-130)) (FHAGSLLVFM) was identified that sensitized targets for lysis and formed stable complexes with H2Db class I molecules. The VP2(121-130) peptide sensitized target cells for lysis by freshly harvested CNS-IL CTLs at femtomolar concentrations. Despite its relative high level of biological activity, the VP2(121-130) peptide is distinguished from other Db-binding peptides by its lack of an asparagine residue at position five, which had been previously proposed to be a requirement for Db-peptide complexing.