The expression of many virulence determinants in Staphylococcus aureus is tightly coordinated generally by global regulatory elements such as accessory gene regulator (agr), staphylococcal accessory regulator and the alternative sigma factor σB. We have compared the two-dimensional (2-D) protein pattern of extracellular protein extracts of wild-type cells with the 2-D patterns of the respective regulatory mutants in order to identify proteins whose amount is influenced by a mutation in agr or sigB. In order to quantify changes in the level of interesting proteins we used the Ettan-fluorescence difference gel electrophoresis technique (Amersham Biosciences). As in most bacteria, the amount of extracellular proteins was strongly regulated and increased mainly in the stationary phase of growth at high cell densities. By comparing the extracellular protein pattern of the RN6390 rsbU strain with that of an isogenic agr mutant RN6911 we show that the level of about 70 protein spots changed in the mutant. To analyze the role of σB in virulence gene expression an RsbU+ (RN6390 RsbU+) derivative was included in this study. The protein pattern of the RsbU+ strain (RN6390 RsbU+) was very similar to that of the Δagr/ΔrsbU mutant strain (RN6911) indicating an opposing effect of agr and rsbU on the expression of the same genes.