Phylogenetic analyses recently found the gene encoding the streptococcal superantigen SSA of Streptococcus pyogenes to occur in several well- differentiated clones comprising 10 (12.5%) of 80 clonal lineages examined. To determine if distinct clonal lineages carried the same ssa coding sequence or harbored a group of allelic variants, ssa was sequenced from 23 S. pyogenes strains representing the 10 clones identified by multilocus enzyme electrophoresis. Three alleles of ssa were found in natural populations of S. pyogenes. ssa-1 and ssa-3 differed by a single synonymous substitution in codon 94; both encoded SSA-1. Each of these alleles was present in phylogenetically diverse clones that had not shared a recent common ancestor. ssa-2 was present in a single clonal lineage. It was identical to ssa-3 at codon 94 but had a nonsynonymous substitution at codon 28 that changed the second amino acid of the mature protein from serine to arginine. This substitution altered the predicted isoelectric point and affected the apparent molecular mass during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Despite sequence variation both upstream of and within the ssa locus, all ssa-positive lineages expressed either SSA-1 or SSA-2. The observed patterns of ssa allele-clone distribution provide evidence for individual incidences of horizontal transfer and recombination of ssa among distinct group A streptococcal lineages. Although the extensive homology of SSA to the staphylococcal superantigen SEB raises the possibility of intergeneric gene transfer, a search for ssa in 68 genetically diverse clones of Staphylococcus aureus did not identify the gene. Moreover, the absence of ssa among 119 representative strains of Lancefield group B, C, or G streptococci suggests that ssa is confined to S. pyogenes.