Staphylococcus aureus produces a family of at least 21 distinct superantigens (SAgs) which include staphylococcal enterotoxins (SEs), staphylococcal enterotoxin-like toxins (SEls), and toxic shock syndrome toxin-1 (TSST-1), and contribute to disease pathogenesis via modulation of the host immune response. Specific SAgs have been shown to cause toxinoses such as staphylococcal food poisoning and toxic shock syndrome, and have been implicated in immunological disorders such as rheumatoid arthritis, psoriasis, and Kawasaki syndrome. However the role of SAgs in disease pathogenesis, in general, is poorly understood. S. aureus is a common cause of bovine mastitis. Analysis of the genome sequence of the bovine strain RF122 revealed genes encoding bovine variants of characterized SAgs, TSST-1, SElL, SEC, SEG, SEI, SElU, SElO, SElN and a truncated form of SElM. In addition we identified 3 genes with sequence homology to characterized SAgs, which are predicted to encode novel SAgs, SElX, SElY and SElZ. Expression of all 11 predicted SAg genes was detected in vitro, including several with growth phase-dependent expression. Characterization of a novel SAg, SElX which is encoded in the core genome of 94% of phylogenetically diverse S. aureus strains from human and animal infections was carried out. In addition to its superantigenic properties, SElX has a unique predicted structure characterized by a truncated SAg B domain. At least 14 different alleles of the selx gene were identified among the common human and animal pathogenic clones, and evidence for assortive recombination of selx alleles between distinct clonal lineages was discovered. SElX was expressed by representative human, bovine and ovine strains in vitro, in a growth phase dependent manner, and during human, bovine and ovine infections, consistent with a broad role in the pathogenesis of different S. aureus diseases in multiple hosts. SElX produced by bovine- and ovine-specialized S. aureus strains had 10-fold greater mitogenic activity and a distinct V activation profile for bovine lymphocytes compared to SElX made by the human strain USA300 indicating functional diversification of selx alleles from different hosts. This is the first description of a core-genome encoded SAg of S. aureus. The discovery that the great majority of S. aureus clinical isolates have superantigenic capacity has important implications for our understanding of staphylococcal disease pathogenesis. To investigate the role of SAgs in disease pathogenesis, a SAg-deficient strain of S. aureus, RF122-8 was constructed by sequential allele replacement. RF122-encoded SAg genes were cloned into the pALC2073 plasmid, which has an inducible promoter allowing controlled expression in the SAg-deficient strain RF122-8. These constructs allowed us to determine that TSST-1bov, SElLbov, SECbov, SElNbov, SEIbov and novel SAgs, SElXbov and SElYbov were mitogenic for bovine Tcells, and stimulated T-cell receptor variable (TRBV) sub-family-specific activation. Preliminary experimental intra-mammary infections of dairy cows revealed that clinical symptoms were similar during infection with wild type RF122 and SAg-deficient strains, including high somatic cell counts (6 LogSCC), and elevated body temperature (106 °F). However a higher infectious dose was required to establish infection with the SAg-deficient strain RF122-8 in comparison to the wild type, RF122 indicating an attenuation of virulence. Overall, these data provide broad new insights into the importance of SAgs in staphylococcal disease pathogenesis.