Epidemiologie and molecular population genetic analyses support a role tor superantigens (SAg) in the pathogenesis of severe streptococcal infections. To better understand how variations in SAg structure influence immunomodulatory activity, we have investigated the biochemical and functional properties of two naturally-occurring allelic variants of the streptococcal SAg SSA that differ by a single amino acid substitution at position 2. Mass spectrometry revealed SSA allelic variants from both recombinant (E. coli) and native (S. pyogenes) sources to have significantly larger molecular masses than predicted by primary sequence alone and provided evidence that the proteins were post-translationally modified by the addition of biochemical moieties, a phenomenon that has not been described for related SAg. Furthermore, the molecular masses of the native and recombinant SAg were not the same; SSA was differentially post-translationally modified by distinct bacterial species. Surprisingly, the substitution of E. coli -de pendent processing for that of S. pyogenes altered both protease digestion patterns and Vβ-specificity of SSA. Thus, although it is critical to ensure against contaminants in native SAg preparations, recombinant preparations from E. coli may not accurately represent the activity of the native toxin. In addition, the observation that SSA allelic variants differing only at amino acid position 2 also differed in V-specificity supports a role for position 2 in SSA-TCR interactions. That SSA position 2 contributes to Vβ-specificity could not have been predicted from functional or crystallographic studies of other SAg and suggests that SSA may adopt unique interactions with TCR and/or MHC class II molecules. Determining the structural basis for these differences should offer clues as to the manner in which SAg exert their effect on the immune system during infection and may allow design of SAg mutants with specific quantitative and qualitative immunomodulatory properties.