Infections with Streptococcus pyogenes (group A streptococcus) can result in the recently described streptococcal toxic shock syndrome (STSS), which is characterized by rashes, hypotension, multiorgan failure, and a high mortality rate. S. pyogenes isolates associated with STSS usually produce streptococcal pyrogenic exotoxin A (SpeA), a bacterial superantigen capable of stimulating host immune cells. Most of the symptoms of STSS are believed to result from cytokine release by the stimulated cells. To better understand the pathogenesis of STSS, we began studies on the SpeA-immune cell interaction. We generated 20 mutant forms of SpeA1 (SpeA encoded by allele 1), and the mutant toxins were analyzed for mitogenic stimulation of human peripheral blood mononuclear cells, affinity for class II major histocompatibility complex molecules (DQ), and disulfide bond formation. Residues necessary for each of these functions were identified. There are four alleles of speA, and STSS strains usually contain either allele 2 or allele 3. The product of allele 2, SpeA2, had slightly higher affinity for the class II MHC molecule compared with SpeA1 but not significantly greater mitogenic activity. SpeA3, however, was significantly increased in mitogenic activity and affinity for class II MHC compared with SpeA1. Thus, we have evidence that the toxin encoded by some of the highly virulent S. pyogenes STSS-associated isolates is a more active form of SpeA.