Resistance to Theiler's virus-induced demyelination maps genetically to the MHC class I D region and is associated with up-regulation of class I products and the presence of MHC-restricted virus-specific cytotoxic CD8+ T cells in the CNS. To determine the targets of the cytotoxic response, transfected C57SV (Kb, Db) cells expressing LP (including the leader peptide, VP4, VP2, and VP3 coding sequences), VP4 (including the leader peptide and VP4), VP2, VP3, VP1, or RP (including P2 and P3) were generated. CNS-infiltrating lymphocytes obtained from virus-infected B10, B10.K (Kk, Dk), B10.RBF (Kb, Df). B10.RFB3 (Kf, Db), and B10.RBQ (Kb, Dq) mice were used as effectors. Specific cytotoxicity to the capsid proteins encoded in the LP construct, VP2 and VP1, was demonstrated to be H-2Db region restricted and was mediated by CD8+ T cells. No Kb-restricted virus-specific cytotoxicity response was observed. No specific cytotoxic response against RP-encoded proteins was observed in the CNS of B10 mice. Therefore, both VP1 and VP2 are targets for an H-2D-restricted cytotoxic immune response against Theiler's virus infection in the CNS of infected resistant B10 mice.