Theiler's murine encephalomyelitis virus (TMEV), a member of the cardiovirus subfamily of the Picornaviridae, is a natural pathogen of mice. Thirty to 60 days following intracerebral infection with TMEV, susceptible inbred mouse strains develop a chronic, progressive, T cell-mediated inflammatory demyelinating disease of the central nervous system (CNS) characterized by spastic hind limb paralysis and a lifelong persistent CNS virus infection. We have examined the effect of peripheral virus-specific tolerance on the development of demyelinating disease. Treatment of SJL/J mice with TMEV-coupled, ethyl carbodiimide-treated splenocytes either before or after infection with live TMEV prevented the development of clinical disease, including inflammation and demyelination in the CNS. Prevention of clinical disease was paralleled by significant reductions in virus-specific immune responses, including delayed type hypersensitivity and T cell proliferative responses. Tolerance induction resulted in a significant reduction in the absolute numbers of mononuclear cells infiltrating the CNS, particularly the CD4+IL-2R+ T cell subset, 3, 5, and 8 wk postinfection. In contrast, tolerance induction had no effect on the numbers of CD8+IL-2R+ T cells infiltrating the CNS. Treatment with TMEV-coupled splenocytes failed to prevent the development of relapsing experimental autoimmune encephalomyelitis, demonstrating the specificity of in vivo tolerance induction. Prevention of demyelinating disease did not correlate with the increased TMEV-specific Ab responses observed in tolerized mice. These results indicate that induction of immune tolerance to TMEV can down-regulate a chronic immunopathogenic disease directed against virus Ag persisting in the CNS that normally results in a progressive demyelinating disease similar to multiple sclerosis.