<jats:title>ABSTRACT</jats:title> <jats:p> Eight different <jats:italic>Bacillus subtilis</jats:italic> strains and <jats:italic>Bacillus atrophaeus</jats:italic> were found to produce the bacteriocin subtilosin A. On the basis of the subtilosin gene ( <jats:italic>sbo</jats:italic> ) sequences two distinct classes of <jats:italic>B. subtilis</jats:italic> strains were distinguished, and they fell into the two <jats:italic>B. subtilis</jats:italic> subspecies ( <jats:italic>B. subtilis</jats:italic> subsp. <jats:italic>subtilis</jats:italic> and <jats:italic>B. subtilis</jats:italic> subsp. <jats:italic>spizizenii</jats:italic> ). The entire sequence of the subtilosin gene cluster of a <jats:italic>B. subtilis</jats:italic> subsp. <jats:italic>spizizenii</jats:italic> strain, <jats:italic>B. subtilis</jats:italic> ATCC 6633, was determined. This sequence exhibited a high level of homology to the sequence of the <jats:italic>sbo-alb</jats:italic> gene locus of <jats:italic>B. subtilis</jats:italic> 168. By using primer extension analysis the transcriptional start sites of <jats:italic>sbo</jats:italic> in <jats:italic>B. subtilis</jats:italic> strains ATCC 6633 and 168 were found to be 47 and 45 bp upstream of the <jats:italic>sbo</jats:italic> start codon, respectively. Our results provide insight into the incipient evolutionary divergence of the two <jats:italic>B. subtilis</jats:italic> subspecies.