The agr quorum-sensing system in Staphylococcus aureus regulates the expression of virulence factors in response to autoinducing peptides (AIPs). Recent studies suggest that the agr system plays a role in biofilm development, as agr mutants exhibit a high propensity to form biofilms, and cells dispersing from a biofilm have been observed displaying an active agr system. This study shows that repression of the agr system is necessary for biofilm formation, and reactivation of the agr system in established biofilms through AIP addition or glucose depletion triggers detachment. Inhibitory AIP molecules did not induce detachment, and an agr mutant was non-responsive, indicating a dependence on a functional, active agr system for dispersal. Biofilm detachment occurred in multiple S. aureus strains with divergent agr systems, suggesting it is a general S. aureus phenomenon. Importantly, detachment also restored sensitivity of the dispersed cells to the antibiotic rifampicin. Proteinase K inhibited biofilm formation and dispersed established biofilms, suggesting agr-mediated detachment occurred in an ica-independent manner. Consistent with a protease-mediated mechanism, increased levels of serine proteases were detected in detaching biofilm effluents, and the serine protease inhibitor PMSF reduced the degree of agr-mediated detachment. Genetic analysis of a double mutant in the agr-regulated Aur metalloprotease and the SplABCDEF serine proteases showed minimal extracellular protease activity, improved biofilm formation, and a strongly attenuated detachment phenotype. These findings indicate that induction of the agr system in established S. aureus biofilms detaches cells and demonstrate that the dispersal mechanism requires extracellular protease activity.The agr quorum-sensing system in Staphylococcus aureus regulates the expression of virulence factors in response to autoinducing peptides (AIPs). Recent studies suggest that the agr system plays a role in biofilm development, as agr mutants exhibit a high propensity to form biofilms, and cells dispersing from a biofilm have been observed displaying an active agr system. This study shows that repression of the agr system is necessary for biofilm formation, and reactivation of the agr system in established biofilms through AIP addition or glucose depletion triggers detachment. Inhibitory AIP molecules did not induce detachment, and an agr mutant was non-responsive, indicating a dependence on a functional, active agr system for dispersal. Biofilm detachment occurred in multiple S. aureus strains with divergent agr systems, suggesting it is a general S. aureus phenomenon. Importantly, detachment also restored sensitivity of the dispersed cells to the antibiotic rifampicin. Proteinase K inhibited biofilm formation and dispersed established biofilms, suggesting agr-mediated detachment occurred in an ica-independent manner. Consistent with a protease-mediated mechanism, increased levels of serine proteases were detected in detaching biofilm effluents, and the serine protease inhibitor PMSF reduced the degree of agr-mediated detachment. Genetic analysis of a double mutant in the agr-regulated Aur metalloprotease and the SplABCDEF serine proteases showed minimal extracellular protease activity, improved biofilm formation, and a strongly attenuated detachment phenotype. These findings indicate that induction of the agr system in established S. aureus biofilms detaches cells and demonstrate that the dispersal mechanism requires extracellular protease activity.