Theiler's murine encephalomyelitis viruses (TMEV) belong to the Picornaviridae, and are divided into two subgroups. TO subgroup strains produce a persistent demyelinating central nervous system infection in mice, while GDVII subgroup strains cause acute polioencephalomyelitis. We generated three overlapping clones of the genome of DA strain, a member of TO subgroup. Sequence analysis revealed that the genome is 8093 nucleotides long with a poly(A) tail. The 5' noncoding region stretches from nucleotides 1 to 1065 and lacks a poly(C) tract. The open reading frame stretches from 1066 to 7968 and encodes 2301 amino acids. DA strain sequence is more closely related to members of the Cardiovirus genus than to members of other Picornavirus genera. Comparison with sequence of BeAn strain, another TO subgroup strain, showed that the P1 area has the greatest number of differences, while the noncoding regions are more well-conserved. The three overlapping clones will be important in recombinant infectious cDNA studies between strains of both subgroups.