Staphylococcus epidermidis releases factors that activate the HIV-1 long terminal repeat, induce cytokine release, and activate nuclear factor κB in cells of macrophage lineage. The active material had a mass of 34,500 daltons, was inactivated by proteases and partitioned into the phenol layer on hot aqueous phenol extraction, and thus was termed phenol-soluble modulin (PSM). High performance liquid chromatography (HPLC) of crude PSM yielded two peaks of activity designated PSM peak 1 and peak 2. MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectroscopy indicated the presence of two components in peak 1, which were designated PSMα and PSMβ. Peak 2 contained a single component, designated PSMγ. Separation of PSMα and PSMβ in peak 1 could be achieved by a second HPLC procedure. The structure of each component was determined by amino acid sequence analysis and identification and sequencing of their genes. PSMα, PSMβ, and PSMγ were 22-, 44-, and 25-amino acid, respectively, strongly hydrophobic polypeptides. PSMγ was identified as Staphylococcus epidermidis delta toxin, whereas PSMα and PSMβ exhibited more distant homology to previously described staphylococcal toxins. They appeared to exist as a complex or aggregate with activity greater than the component parts. The properties of the S. epidermidis PSMs suggest that they may contribute to the systemic manifestations of Gram-positive sepsis.