SigB, a newly discovered alternative sigma factor of Staphylococcus aureus, has been shown to play an important role in stress responses and the regulation of virulence factors. The rsbW (orf159) gene is immediately upstream of sigB. Its gene product is homologous to Bacillus subtilis RsbW which under appropriate conditions binds to B. subtilis SigB and functions as an anti-sigma factor or negative posttranslational regulator. To define the function of S. aureus RsbW, both the S. aureus SigB and RsbW proteins were expressed in Escherichia coli and purified. Cross-linking experiments with these purified proteins revealed that RsbW was capable of specific binding to SigB. In an in vitro transcription runoff assay, RsbW prevented SigB-directed transcription from the sar P3 promoter, a known SigB-dependent promoter, and the inhibitory activity of RsbW was found to be concentration dependent. We also identified SigB promoter consensus sequences upstream of the genes encoding alkaline shock protein 23 and coagulase and have demonstrated SigB and RsbW dependence for the promoters in vitro. These results show that RsbW is a protein sequestering anti-sigma factor of S. aureus SigB and suggest that SigB activity in S. aureus is regulated posttranslationally.