Theiler's murine encephalomyelitis virus (TMEV) causes a demyelinating disease similar to multiple sclerosis in the central nervous system (CNS) of susceptible SJL/J mice. Immune responses to TMEV contribute to viral clearance as well as to demyelination. We constructed recombinant vaccinia viruses (VV) that encode each or all of the capsid proteins (VV(VP1), VV(VP2), VV(VP3), VV(VP4), and VV(all)) or non-structural proteins (VV(P2), VV(P2P3), and VV(3'P3)) of the Daniels strain of TMEV. To determine the role of each of the coding regions of TMEV in vivo, we immunized SJL/J mice with each recombinant VV, with or without subsequent TMEV infection. The groups of mice were compared clinically, immunologically, and histologically. No mice immunized with any recombinant VV without subsequent TMEV infection developed demyelination. However, antibody responses to TMEV were detected in mice immunized with VV(all). In addition, in some mice, VV(P2) immunization induced mild meningitis. VV(VP3) or VV(VP4) immunization of mice prior to TMEV infection ameliorated TMEV-induced pathology or clinical signs of disease. The beneficial effect of VP4 immunization was also seen through DNA immunization with a plasmid encoding VP4 and leader prior to TMEV infection. Therefore, vaccination against not only surface capsid proteins (VV(VP3) and VV(all)) but also non-surface capsid protein (VV(VP4)), and non-structural proteins (VV(P2)) can elicit immune responses to virus or modulate subsequent viral-induced CNS disease.