Abstract Staphylococcus aureus is one of the major pathogens that can cause staphylococcal infection and food poisoning. There are five major classical types of staphylococcal enterotoxins (SEs): SEA, SEB, SEC, SED, and SEE, as well as new SEs or SE-like superantigens (SAgs), such as SEG to SEU. Since many S. aureus strains harbor more than one SE gene and identification of SEs involved in food poisoning cases is time consuming, we developed a chromogenic macroarray method that allows convenient and simultaneous detection of classical SE genes and a new SE gene (seg), which is phylogenetically highly related to seb and sec. Two sets of degenerated primers labeled with biotin were used to co-amplify all SE genes in S. aureus strains through the polymerase chain reaction (PCR). Afterwards, these biotin-labeled PCR products were hybridized with SE gene-specific probes spotted on the nitrocellulose membrane. When this macroarray was used to detect enterotoxingenic S. aureus in milk or beef homogenate containing 10(0)-10(4) target cells per milliliter or gram of the sample, all six enterotoxin genes could be identified after a 12-hour enrichment step. This macroarray offers clinical and food inspection laboratories a rapid and economical visual method to detect common enterotoxigenic S. aureus strains.