The nucleotide sequence of the hemagglutinin (ha) genes and the transcriptional regulator botR gene were determined in type A Clostridium botulinum strain 62A, and the complete nucleotide sequence of the botulinum neurotoxin (BoNT) gene cluster was determined in strain Hall A-hyper. Comparison of the BoNT/A gene clusters revealed only two nucleotide differences between the two strains. The nucleotide sequences of the regions flanking the BoNT clusters were also determined in strains 62A, Hall A-hyper, and type A(B) strain NCTC 2916. The regions upstream of the BoNT/A clusters in the type A strains shared marked homology with the region upstream of the silent BoNT/B cluster in the A(B) strain, indicating a similar evolutionary origin. The region downstream of the BoNT/A cluster in type A strains encodes putative insertion sequence (IS) elements with multiple internal mutations. These IS elements may have played a role in neurotoxin gene transfer within the host genome and to other Clostridium species.