The complete nucleotide sequence of the genome of the coxsackievirus B1, a human enterovirus that belongs to the Picornaviridae, was determined by using molecular cloning and rapid sequence analysis techniques. Sequence analysis of the cloned cDNAs revealed that the virion RNA was 7389 nucleotides long and polyadenylylated at the 3' terminus. Similar to other picornavirus genomes, a single large open reading frame was identified. The translated sequence starts at nucleotide position 742 and ends at 7287 of the genome. Thus, the viral polyprotein should consist of 2182 amino acids. When the predicted amino acid sequence of the viral polyprotein was compared with those of other human enteroviruses such as polioviruses, a striking sequence homology was observed, especially in viral proteins 1B, 2C, and 3D. This allowed us to predict precise map locations of the viral structural and nonstructural proteins on the genome, although two proteolytic processing sites, between 1D and 2A and between 2B and 2C, were obscure. The result presented here implied important information with respect to the genetical variation of human enteroviruses.