The three-dimensional structure of an unglycosylated T cell antigen receptor (TCR) β chain has recently been determined to 1.7 Å resolution. To investigate whether this soluble β chain (murine Vβ8.2Jβ2.ICβ1) retains superantigen (SAG)-binding activity, we measured its affinity for various bacterial SAGS in the absence of MHC class II molecules. Dissociation constants (KDs) were determined using two independent techniques: surface plasmon resonance detection and sedimentation equilibrium. Specific binding was demonstrated to staphylococcal enterotoxins (SEs) B, C1, C2, and C3 and to streptococcal pyrogenic exotoxin A (SPEA), consistent with the known proliferative effects of these SAGs on T cells expressing Vβ8.2. In contrast, SEA, which does not stimulate Vβ8.2-bearing cells, does not bind the recombinant β chain. Binding of the β chain to SAGs was characterized by extremely fast dissociation rates (>0.1 s-1), similar to those reported for certain leukocyte adhesion molecules. Whereas the β chain bound SEC1, 2, and 3 with KDs of 0.9-2.5 µM, the corresponding value for SEB was ~140 µM. The much weaker binding to SEB than to SEC1, 2, or 3 was surprising, especially since SEB was found to actually be 3- to 10-fold more effective, on a molar basis, than the other toxins in stimulating the parental T cell hybridoma. We interpret these results in terms ofthe ability of SEC to activate T cells independently of MHC, in contrast to SEB. We have also measured SE binding to the glycosylated form of the β chain and found that carbohydrate apparently does not contribute to recognition, even though the N-linked glycosylation sites at Vβ8.2 residues Asn24 and Asn74 are at or near the putative SAG-binding site. This result, along with the structural basis for the Vβ specificity of SEs, are discussed in relation to the crystal structure of the unglycosylated β chain.