It is well established that the bacterial superantigens bind to both TCR beta-chain and, with moderate affinity, to MHC class II molecules. Class II-bearing cells bind the superantigen and present the superantigen to T cells expressing certain TCR beta-chain variable region alleles. We have observed that the superantigen staphylococcal enterotoxin (SE) B binds to COS-1, an African green monkey kidney fibroblast-like cell line. This cell line fails to express class II and the binding of SEB is saturable. Scatchard analysis of radiolabeled ligand binding data reveals a binding affinity for COS of 51 nM, while binding to DR1-transfected L cells is measured at 150 nM. Further analysis shows that SEB bound to COS cells, unlike SEB bound to DR1-bearing L cells, is not recognized by T cells in the presence or absence of accessory cytokines or anti-CD28. Studies carried out with chemical cross-linking agents show that radiolabeled SEB associates with a membrane protein of approximately 85 kDa. These studies suggest that the alternative superantigen binding molecule (p85) binds SEB with an affinity roughly equivalent to SEB binding by class II molecules, but at a site which does not permit recognition by the TCR.