The association between staphylococcal enterotoxin (SE) B and toxic shock syndrome (TSS) toxin 1 (TSST-1) in menstrual and nonmenstrual TSS was examined. Production of SEB and TSST-1 in the culture supernatants from 344 Staphylococcus aureus isolates was quantitated by noncompetitive ELISA (lower detection limit, 0.5 ng/mL for both assays). In the ELISA for SEB, cross-reactive antibodies to SEC were removed by absorption in a biotin-streptavidin-agarose column (SEC1 at concentrations to 100,000 ng/mL could not be detected). Among TSST-1-negative isolates, SEB production in nonmenstrual TSS isolates (8/13, 62%) was more prevalent than that in either menstrual TSS isolates (1/4; P = .3), non-TSS infections (15/103, 15%; P less than .001), or asymptomatic carriers (14/105, 13%; P less than .001). Expression of SEB and TSST-1 was usually mutually exclusive (only 1.5% of 344 isolates produced both SEB and TSST-1). Multilocus enzyme electrophoretic typing (ET) suggested a clonal origin for both SEB-(55% of ET-5) and TSST-1 (76% of ET-19)-producing isolates. Thus, SEB may be an important cause in TSS where TSST-1 is not elaborated, especially in nonmenstrual TSS isolates.