Toxic shock syndrome toxin 1 (TSST-1) and staphylococcal enterotoxin B (SEB) are two examples of variable genetic traits produced by Staphylococcus aureus. Regarding phenotypes, most investigations have reported that SEB and TSST-1 are not coproduced in S. aureus. However, it is unclear whether genotypically SEB (entB) and TSST-1 (tst) genes are found within the same isolate. Gene probes specific for entB and tst showed that both genes were present within 8 TSST-1+ isolates analyzed. However, SEB was not produced by any of these isolates. In contrast, none of 9 SEB-producing S. aureus isolates contained the TSST-1 genetic element DNA. Hybridization of restriction fragments with TSST-1 and SEB genetic element probes and tryptophan auxotyping suggested that tst and entB are located in close proximity on the chromosome. The entB locus may be a preferred site of insertion for the TSST-1 genetic element, and such insertion may interfere with the expression of SEB.