Theiler's murine encephalomyelitis viruses (TMEV) are picornaviruses that produce enteric and neurological diseases in mice. Subgroup TO strains of TMEV cause persistent infections with demyelination, while subgroup GDVII strains neither persist nor demyelinate. We produced neutralizing monoclonal antibodies (mAbs) to clarify the mechanisms of persistence and demyelination. Some of the neutralizing mAbs reacted with isolated VP1 on Western blots, while others were conformation specific. The neutralization site for the former TMEV mAbs was on the VP1 trypsin cleavage site of the intact virion. The neutralization site for the conformation-specific mAbs was distinct and was not affected by trypsin. Trypsin treatment of subgroup TO strains increased their infectivity for L cells, whereas the infectivity of subgroup GDVII strains was decreased by trypsin treatment. Subpopulations of virus in subgroup TO-infected tissue culture cells and in infected mouse brain homogenates contained VP1-cleaved virus; this VP1-cleaved virus gave rise to a large persistent fraction in neutralization tests when it was reacted with VP1-specific mAbs. These findings have implications regarding the pathogenesis of subgroup TO demyelinating disease. TMEV VP1 cleavage may be important for virus persistence because of disruption of a major neutralization epitope. The change in virus surface structure caused by VP1 cleavage may affect cell binding and lead to altered cytotropism. Immunocytes, which have been implicated in subgroup TO demyelination, may provide a source for proteases for VP1 cleavage.