Sequence analysis of VP1 in the DA strain of Theiler's murine encephalomyelitis viruses (TMEV) showed that 13 of the first 23 N-terminal amino acids were identical to those in the corresponding protein of encephalomyocarditis virus. There was little similarity to the corresponding VP1 sequences of poliovirus types 1, 2 and 3, coxsackievirus B3, human rhinoviruses 2 and 14, human hepatitis A virus or foot-and-mouth disease virus. These results, as well as serological relationships detected by immunoblotting, suggest that the TMEV are more closely related to the cardioviruses than to the enteroviruses with which they are presently classified. This newly recognized relationship suggests potential for recombinant infectious cDNA studies between TMEV and cardioviruses.