The hallmark of T cell responses to staphylococcal enterotoxins (SE) and other super-Ag is a selective stimulation of cells expressing particular TCR- Vβ segments. Our previous studies suggested that the disulfide loop in SE is critical for their interaction with the TCR. To investigate this concept in further detail we constructed disulfide loop mutants of staphylococcal enterotoxin A (SEA), and examined these altered toxins for mitogenicity, class II MHC binding, and Vβ specificity. We found that substitutions of either Cys-96 or Cys-106 decreased mitogenicity by 100-fold without significantly affecting class II binding or resistance of the molecule to proteolysis. Several mutants lost the capacity to stimulate Vβ11+ cells, except a Cys-106→Gln mutant for which Vβ11-stimulatory activity was increased. By contrast, mutants containing Cys→Ala substitutions acquired the capacity to stimulate Vβ6+ cells. Despite these effects on Vβ specificity, all mutants retained the predominant preference of SEA for Vβ3+ cells. Neither exchange of regions flanking the loop in SEA with corresponding residues in SEB, nor conversion of the entire loop region of SEA to that of SEE, were associated with transfers of Vβ specificity. Our results suggest that the disulfide loop in SEA contributes to toxin avidity for the TCR, rather than specificity for particular Vβ.