Staphylococcus aureus enterotoxin D is one of the serotypes most commonly associated with food poisoning. Further characterization of the enterotoxin D-encoding plasmid revealed the presence of an open reading frame which encodes a previously unidentified enterotoxin, designated staphylococcal enterotoxin J (SEJ). SEJ is a protein of 269 amino acid residues which has substantial sequence similarity to the staphylococcal A, E, D family of enterotoxins. The enterotoxin D and J open reading frames are transcribed in opposite directions and are separated by an 895 nucleotide intergenic region which contains a perfect inverted repeat, with each arm of the repeat having a length of 21 nucleotides. Chloramphenicol acetyl transferase (cat) transcriptional fusions were used to quantify expression from the enterotoxin gene promoters. Both enterotoxin genes are expressed in S. aureus. However, only sed is regulated by the agr virulence gene signal transduction pathway. Western blot analyses utilizing anti-enterotoxin antisera have confirmed the results obtained with the cat reporter system. PCR amplification studies suggest that the sej determinant may be present on all sed-encoding plasmids.