Crystallographic studies reveal even distantly related bacterial superantigens (SAg) to adopt a common structural topology. Mutational analyses confirm that this shared folding pattern often confers a conserved function to analogous residues in different SAg, albeit with specificities for particular TCR or MHC class II molecules. It was thus surprising that the streptococcal SAg SSA differed from related SAg in the location of its Vβ-determining residues. Because it seemed unlikely that SSA would deviate significantly from an SAg-like topology, we hypothesized that variations in SSA Vβ-determining regions might result from differences in SSA-MHC class II interactions relative to other SAg during SSA presentation to the TCR. Comparison of the DR1-binding properties of SSA with its closest homologue SEB found different amino acid positions within SAg primary sequences to contribute to SSA-DR1 and SEB-DR1 interactions, and suggested that SSA bound DR1 with an altered orientation relative to SEB. The common involvement of DR1 α39K, however, predicted that the two SAg bound overlapping sites on DR1. Nevertheless, SSA and SEB did not effectively cross-compete for DR1 binding and had opposite patterns of DR1-binding affinity in the presence of distinct DR1-expressing cell lines. The data thus suggest that SSA and SEB bind not only with different orientations on DR1, but may bind preferentially to distinct DR1 subsets delineated by cell-specific factors. Differences in orientation of SSA on DR1 and/or interaction of SSA with particular DR1 subsets may explain why unconventional regions influence SSA TCR Vβ specificity.