We have previously shown that sequence 121-149 of the staphylococcal enterotoxin superantigen SEA plays an important role in superantigen function. A synthetic peptide of this region, SEA(121-149), blocks SEA binding to class II MHC molecules and induces interleukin-1 and tumor necrosis factor production in monocytes. In this study, we further emphasize the structural and functional significance of this region of SEA by showing that the SEA(121-149) peptide induces T cell proliferation in a manner similar to that of SEA. SEA(121-149) reacted with antibodies produced to SEA, and the SEA(121-149) specific antibodies neutralized SEA mitogenic activity. A tetrameric form of SEA(121-149) showed increased binding to antibodies and enhanced T cell activation, consistent with the greater avidity associated with increased valency. These data suggest that the internal domain of SEA corresponding to residues 121-149 plays an important role in superantigen activity.