The recently described staphylococcal enterotoxins (SE) G and I were originally identified in two separate strains of Staphylococcus aureus. We have previously shown that the corresponding genes seg and sei are present in S. aureus in tandem orientation, on a 3.2-kb DNA fragment (Jarraud, J. et al. 1999. J. Clin. Microbiol. 37:2446-2449). Sequence analysis of seg-sei intergenic DNA and flanking regions revealed three enterotoxin-like open reading frames related to seg and sei, designated sek, sel, and sem, and two pseudogenes, psi ent1 and psi ent2. RT-PCR analysis showed that all these genes, including seg and sei, belong to an operon, designated the enterotoxin gene cluster (egc). Recombinant SEG, SEI, SEK, SEL, and SEM showed superantigen activity, each with a specific V beta pattern. Distribution studies of genes encoding superantigens in clinical S. aureus isolates showed that most strains harbored such genes and in particular the enterotoxin gene cluster, whatever the disease they caused. Phylogenetic analysis of enterotoxin genes indicated that they all potentially derived from this cluster, identifying egc as a putative nursery of enterotoxin genes.