The chromosomal location of the enterotoxin B gene in Staphylococcus aureus is unknown. Southern hybridization analysis of the chromosomal DNA from several enterotoxin B (SEB)-producing strains has shown that at least 26.8 kilobases (kb) of DNA is associated with the enterotoxin B gene (entB). We have found that one end of the entB element is located approximately 1.5 kb downstream of the entB gene. The chromosomal region adjacent to this end of the entB element was found to be homologous in several SEB-producing (SEB+) and SEB-nonproducing (SEB-) S. aureus strains. The chromosomes of all the SEB+ strains studied were homologous for at least 24 kb upstream of the entB gene. Some naturally occurring SEB- strains lacked the entire entB element, while others showed variable homology to the region upstream of the entB gene. These data suggest that the entB gene is part of a discrete genetic element that is at least 26.8 kb in size.