The nucleotide sequence of coxsackievirus B6 (CVB6) has been determined, and the nucleotides encoding the 5' nontranslated region (5' NTR) and virion polypeptides (VP4, 2, 3 and 1) were compared with other serotype CVBs. An Unweighted Pair-Group Method Analysis (UPGMA) of phylogenetic trees indicated that the 5' NTR of CVB6 locates on an independent branch from the other CVBs. The tree based on the amino acid sequences showed that CVB6 has close correlation with CVB4 in the VP4 and VP2 regions, with CVB1 and CVB5 in the VP3 region, and with CVB5 in the VP1 region. Amino acid sequences of variable regions within the VP2, VP3, and VP1 of CVB6 were unique among CVBs. Thus, by comparison of the nucleotide and amino acid sequences of these variable regions, CVB6 can be easily distinguished from other serotypes. In addition, serine, instead of glycine, was found to locate at the amino-terminus of the VP1 region of CVB6, indicating that CVB6 has a unique cleavage site (i.e., glutamine/serine instead of glutamine/glycine) for proteinase 3C of Picornaviridae.