Staphylococcus epidermidis is the most frequent cause of nosocomial sepsis and catheter-related infections, in which biofilm formation is considered to be the main virulence mechanism. Quorum-sensing systems have been recognized as important regulators of virulence and biofilm formation in many bacteria. There is a single quorum-sensing system in S. epidermidis encoded by the agr operon. To investigate quorum-sensing control of biofilm formation, we constructed an agr deletion mutant, assayed for the different stages of biofilm formation, and determined agr-dependent regulation of biofilm factors. The agr mutant showed increased biofilm formation, primary attachment, and expression of the autolysin AtlE, but lacked δ-toxin production. However, the level of polysaccharide intercellular adhesin expression was equivalent to the isogenic wild-type strain. In contrast to AtlE, which is known to influence primary attachment, δ-toxin appeared to exert its effect on attachment to polystyrene during later stages of biofilm formation. Importantly, addition of cross-inhibiting pheromones mimicked an agr mutation and significantly enhanced biofilm formation, which suggests that care should be used when treating S. epidermidis infections with cross-inhibiting peptides. Our data demonstrate the importance of quorum sensing in the establishment of a biofilm in this critical human pathogen