Concentrated supernates of cultures of 98 strains of Staphylococcus aureus were screened for the production of epidermolytic toxin by (1) biological tests in 3-day-old mice, (2) double-diffusion precipitation tests against specific antiserum, and (3) the appearance of characteristic protein bands on thin-layer-gel isoelectric focusing. Positive results were obtained in all three of these tests with supernates from 11 of these cultures; the same 11 strains, and no others, produced epidermal splitting when newborn mice were challenged with viable organisms. Of the 14 phage-group II strains included in the survey, eight (58%) produced epidermolytic toxin. Three toxinogenic strains belonged to phage groups other than group II. A radial-immunodiffusion test employing antiserum to purified epidermolytic toxin proved satisfactory for measuring amounts of epidermolytic toxin in excess of 200 mug per ml. The results of immunodiffusion tests indicated that six of the 11 positive strains produced two serologically distinct forms of epidermolytic toxin and that the remainder produced only one of these. A striking correlation was observed between the presence of toxin of serotypes i and ii and the occurrence of protein bands i and ii in thin-layer isoelectric-focusing gels. These tests should facilitate the qualitative and quantitative assessment of the production of different serotypes of epidermolytic toxin by S. aureus in future surveys of strains isolated from toxic epidermal necrolysis of Ritter's type and impetigo. The two forms of epidermolytic toxin previously designated ETA (pI=7-0) and ETB (pI=6-0) were detected by preparative isoelectric focusing of sero-type-i toxin. Evidence suggests that studies of the effect of heat should provide a means of investigating the relationship between the different molecular forms of epidermolytic toxin.