This study compared the promoter activation profiles of the 2 major transcripts of the Staphylococcus aureus global regulon, agr (RNAII and RNAIII). In vitro, RNAIII activation temporally followed RNAII activation and was absent in agr mutants. In experimental endocarditis, maximal RNAII activation in vegetations occurred early, followed by progressive increases in RNAIII activation (P<.05; 2 vs. 48 h); this paralleled significant increases in vegetation bacterial densities over time (P<.05; 2 and 6 vs. 48 h). At 48 h of infection, S. aureus densities in vegetations were significantly higher than those in kidney or spleen tissue (P<.05), paralleling a significantly greater RNAIII activation profile in vegetations than in the latter tissues (P<.05). Of importance, RNAIII activation was observed in vegetations in 2 agr mutants. These data demonstrate that RNAIII activation in vivo is time and cell density dependent, may be tissue specific, and can occur through RNAII-dependent and -independent mechanisms