The E2 strain of coxsackie B4 virus (CB4), which is of human origin, can induce a diabetes-like syndrome in mice. The cDNA of the genome of the E2 strain was cloned and sequenced. The E2 viral genome was found to comprise 7,396 bases, which appear to encode a polyprotein of 2,183 amino acids with an overall similarity of 94.91% to nondiabetogenic CB4 prototype JBV strain. The E2 genome is organized like other enteroviruses. It has a 5' noncoding region of 744 nucleotides, a single long open translational reading frame starting at nucleotide 745 and extending to nucleotide 7293, a 3' noncoding region of 100 nucleotides, and a poly (A) tract. Genomic sequence comparison of the E2 and JBV strains showed 1,369 nucleotide substitutions in the genome of the E2 strain, most of which are single and silent. There were 111 resultant amino acid changes arising from some of these substitutions, including 82 amino acid changes in the noncapsid proteins, and 29 amino acid changes in the capsid proteins VP1, VP2, VP3, and VP4, which showed 11, 13, 4, and 1 substitution(s), respectively. Noncapsid protein P2-C showed eight amino acid substitutions. On the basis of the sequence comparison of E2 and JBV strains of CB4, we suggest that some of the amino acid changes in the capsid and noncapsid proteins of the E2 strain may be involved in the determination of its diabetogenicity.