Staphylococcal superantigens (SAgs) comprise a large family of exotoxins produced by Staphylococcus aureus strains. These exotoxins are important in a variety of serious human diseases, including menstrual and nonmenstrual toxic shock syndrome (TSS), staphylococcal pneumonia and infective endocarditis, and recently described staphylococcal purpura fulminans and extreme pyrexia syndrome. In addition, these SAg exotoxins are being increasingly recognized for their possible roles in many other human diseases, such as atopic dermatitis, Kawasaki syndrome, nasal polyposis, and certain autoimmune disorders. To clarify the full spectrum of human diseases caused by staphylococcal SAgs, it is necessary to have assays for them. At present there are 23 characterized, serologically distinct SAgs made by S. aureus: TSS toxin-1(TSST-1); staphylococcal enterotoxins (SEs) A, B (multiple variant forms exist), C (multiple minor variant forms exist), D, E, and G; and SE-like H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, and X. The most straightforward way to analyze S. aureus strains for SAgs is through polymerase chain reaction for their genes; we provide here our method for this analysis. Although it would be ideal to confirm that all of the same SAgs are produced by S. aureus strains that have the genes, antibody reagents for SAg detection are only available for TSST-1; SEs A-E and G; and enterotoxin-like proteins H, I, Q, and X. We provide a Western immunoblot procedure that allows in vitro quantification of these SAgs.