A cDNA copy covering two-thirds of the coxsackievirus B3 genomewas cloned in the PstI site of the pBR322 vector. A nucleotidesequence containing the gene for the viral replicase and the3' noncoding region of the coxsackievirus B3 genome was determined.The predicted amino acid sequence of the coxsackievirus B3 replicasewas shown to be remarkably similar to that of the poliovirus1 replicase. The 3' noncoding region, in contrast, was onlyweakly homologous to the poliovirus 1 sequence but showed aclose relationship to the sequence of swine vesicular diseasevirus, a variant of coxsackievirus B5. A 13-nucleotide-longsegment located near the polyadenylic acid junction is conservedin several members of the enterovirus group and may thus servean important function during replication of viral RNA.