The cluster of genes encoding components of the botulinum neurotoxin (BoNT) complex was mapped in proteolytic (group I) Clostridium botulinum strains encoding BoNT types A, B, and F. Two different arrangements of genes were found: type A strain 62A and type B strain NCTC 7273 have similar organizations of genes encoding BoNT, the nontoxic nonhemagglutinin component (NTNH), hemagglutinin components, and P-21; type F strain Langeland has genes encoding BoNT, NTNH, and P-21, and a previously unidentified open reading frame encoding a protein of 416 amino acids. A group of type A strains typified by infant strain Kyoto-F, which is unlike type A strain 62A, lacks genes for hemagglutinin components and exhibits an organization similar to that of type F. Sequencing and pairwise analysis revealed the presence of possible chimeric sequences in some NTNH genes of proteolytic C. botulinum. Discordance in genealogical trees derived from different regions of the NTNH genes was observed which could be symptomatic of recombination and which may indicate that the NTNH gene represents a hot spot for such events within the cluster of genes encoding the BoNT complex. It is also evident that the phylogenetics of the NTNH gene, which is linked to the gene encoding BoNT, does not mirror the evolutionary history of the BoNT, upon which the C. botulinum species complex is defined and subdivided.