To analyse the intratypic genome variability of coxsackievirus B1, 17 coxsackievirus B1 isolates were collected over a period of 10 years. Nucleotide sequences of the 2A coding region of the various coxsackievirus B1 isolates and known sequences of other enteroviruses were compared. The maximum diversity observed within the entire group of coxsackievirus B1 isolates was 25%. Comparison of deduced amino acid sequences revealed a maximum diversity of 5%. Phylogenetic analysis demonstrates a close genetic relationship between these clinical isolates and the other different coxsackievirus B serotypes. Further analysis of nucleotide and amino acid sequences of the 2A region of known enteroviruses demonstrated that the genus enterovirus can be subdivided into a coxsackievirus B-like group, including the coxsackie B viruses, coxsackievirus A9, echovirus 11 and swine vesicular disease virus, and a poliovirus-like group including the polioviruses 1 to 3, and the coxsackieviruses A21 and A24. Enterovirus type 70 and bovine enterovirus represent distinct groups. The 2A coding region of coxsackievirus B1 strains cannot be distinguished from that of other members of the coxsackievirus B-like group. Although conservation of the overall amino acid sequence was almost limited to residues essential for proposed enzymatic activity, the predicted secondary structures were well conserved within the genus enterovirus.