Phenol-soluble modulin (PSM) is a peptide complex produced by the nosocomial pathogen Staphylococcus epidermidis that has a strong capacity to activate the human innate immune response. We developed a novel method based on liquid chromatography-mass spectrometry (LC-MS) to quantify the production of the individual PSM components. Each PSM peptide was abundant in most of the 76 S epidermidis strains tested. Importantly, none of the PSM components were secreted by an agr mutant strain, indicating that PSM synthesis is regulated strictly by the agr quorum-sensing system. Furthermore, the agr mutant strain failed to elicit production of TNFalpha by human myeloid cells and induced significantly less neutrophil chemotaxis compared with the wild-type strain. Thus, quorum-sensing in S. epidermidis dramatically influenced activation of human host defence. We propose that an agr quorum-sensing mechanism facilitates growth and survival in infected hosts by adapting production of the pro-inflammatory PSMs to the stage of infection.