Production of protein A (SpA) in Staphylococcus aureus is controlled by several global regulators including agr (RNAIII), sarA, sarS, sarT, rot and mgrA, which appear to form a regulatory network. SarS, which is an activator of spa, seems to be a key regulator in this network. Previous studies have shown that expression of sarS is upregulated in agr, sarA and mgrA mutants, resulting in increased spa expression. In an agr mutant, upregulation of sarS and spa required rot and sarT. In this study regulatory proteins binding to spa and sarS promoter fragments coupled to magnetic beads were identified by MALDI-TOF-MS. Expression of sarS and spa in mutants deficient for one, two or three of the identified regulators and in mutants over-expressing selected regulators was analyzed by Northern and Western blotting. Binding and transcription analysis indicated that SarA represses sarS by direct binding to the promoter, and that stimulation of sarS by sarT was rot-dependent. We also found that MgrA bound to the sarS promoter and was required for expression of both sarS and spa in an agr mutant. However, mgrA and rot were not required for stimulation of sarS in the absence of SarA, suggesting that these regulators, together with SarT, counteract the repressive activity of SarA. Our data indicate that SarS competes with SarA for binding to the spa promoter. This is consistent with the requirement for sarS in spa transcription even in the absence of the repressor, SarA. We also demonstrated binding of MgrA and Rot to the spa promoter, and that rot could stimulate spa transcription even in a sarS mutant. A provisional model for spa regulation involving agr, sarA, sarS, sarT, rot and mgrA is presented.