Several serious diseases are caused by biofilm-associated Staphylococcus aureus, infections in which the accessory gene regulator (agr) quorum-sensing system is thought to play an important role. We studied the contribution of agr to biofilm development, and we examined agr-dependent transcription in biofilms. Under some conditions, disruption of agr expression had no discernible influence on biofilm formation, while under others it either inhibited or enhanced biofilm formation. Under those conditions where agr expression enhanced biofilm formation, biofilms of an agr signaling mutant were particularly sensitive to rifampin but not to oxacillin. Time lapse confocal scanning laser microscopy showed that, similar to the expression of an agr-independent fluorescent reporter, biofilm expression of an agr-dependent reporter was in patches within cell clusters and oscillated with time. In some cases, loss of fluorescence appeared to coincide with detachment of cells from the biofilm. Our studies indicate that the role of agr expression in biofilm development and behavior depends on environmental conditions. We also suggest that detachment of cells expressing agr from biofilms may have important clinical implications.