The goal of this study was to investigate the molecular interaction between superantigens and the T-cell receptor (TCR). Using a quantitative polymerase chain reaction (PCR) to assess T-cell proliferation profiles, we found that SEB, SEC1, SEC2 and SEC3 expanded human T cells bearing V beta 3, V beta 12, V beta 13.2, V beta 14, V beta 15, V beta 17 and V beta 20. SEC2 and SEC3 have the additional ability to expand T cells bearing V beta 13.1, and their expansion of V beta 3 was markedly reduced compared to SEB and SEC1. Based on the activity of SEC1 mutants containing single amino acid substitutions, we concluded that the differential abilities of these native toxins to stimulate V beta 3 and V beta 13.1 was determined by the residue in position 26, located in the base of the SEC alpha 3 cavity. The SEC1 mutant, in which Val in position 26 was substituted with the analogous SEC2/SEC3 residue (Tyr), generated a V beta expansion profile that was indistinguishable from those generated by SEC2 and SEC3. Using these findings, the co-ordinates of a recently reported murine TCR beta-chain crystal structure, and other documented information, we propose a compatible molecular model for the interaction of SEC3 with the T-cell receptor. In this model complex, the complementarity-determining regions (CDRs) 1 and 2 and the hypervariable loop 4 of the V beta element contact SEC3 predominantly through residues in the alpha 3 cavity of the toxin. CDR3 of the beta chain is not involved in any toxin contacts. The proposed model not only includes contacts identified in previous mutagenesis studies, but is also consistent with the ability of tyrosine and valine in position 26 to differentially affect the expansion of V beta s 3 and 13.1 by the SEC superantigens.