The expression of antiphagocytic polysaccharide capsules is an important pathogenetic step in establishing Staphylococcus aureus infections. Using a green fluorescent protein reporter gene (gfp) system, we examined the expression and genetic regulation of the cap5 promoter (capsular polysaccharide 5 genes) by two major global regulators of S. aureus (agr and sarA) in vitro and in a rabbit endocarditis model. In vitro, cap5 expression substantially increased during the post-exponential phase in parental, as well assarA mutant constructs. However, cap5 expression was greatly reduced in agr and agr/sarA double mutants. In the endocarditis model, the extent of cap5 expression in vegetations infected with the parental strain was substantially higher than that observed with the agr/sarA double mutants (P<0.05). Similar trends were noted in renal, but not splenic abscesses. Collectively, these data suggest that agr positively regulates cap5 expression both in vitro and in vivo, while the contribution of sarA to cap5 regulation, although modest, is readily discerned in vivo in agr minus background. In addition, the regulation ofcap5 expression by these global regulators may vary in distinct anatomic niches in vivo.