Intracranial infection of susceptible mice with Theiler's virus results in persistent infection and spinal cord demyelination similar to human multiple sclerosis. While central nervous system infiltrating lymphocytes (CNS-ILs) in these mice display no virus-specific CTL activity, the cells were found to be activated killers using a specificity-independent assay. We previously demonstrated that the depletion of T cells in persistently infected mice significantly decreases demyelinating disease. Consequently, we have investigated the killing pathways employed by CNS-ILs that are isolated from persistently infected animals, the relative contribution of CD4 and CD8 cells in the generation of these CTLs, and the reactivity of this cell population to two putative autoantigens in the CNS. In vitro or in vivo manipulation of T cell populations using Abs or genetic knockout strategies demonstrate that the cytotoxic activity is primarily mediated by CD8+ T cells, and that perforin is an important molecule in the effector pathway. Since effector functions in infected mice were not inhibited by the depletion of CD4 cells with mAb but was blocked genetically in CD4 knockout mice, CD4+ T cells appear to play a helper role in the generation of CD8+ CTLs. We found no evidence of autoimmune-mediated demyelination, as the CD8+ CTLs were not reactive to two major myelin autoantigens, myelin basic protein and proteolipid protein. Our finding that CNS-ILs that are isolated from mice susceptible to persistent virus infection are neither specific for virus or myelin autoantigens is consistent with the possibility that CD8+ CTLs mediate CNS damage as a result of nonspecific activation by virus.