The expression of extracellular virulence determinants in Staphylococcus aureus is controlled by a 510-nucleotide RNA molecule (RNAIII) which is a part of the agr system. The agr operon, which encodes a multicomponent signal transduction system, is partially under the influence of an unlinked regulatory locus called sar. The sar locus is composed of three overlapping transcripts, designated sarA (0.56 kb), sarC (0.8 kb), and sarB (1.2 kb), originating from the P1, P3, and P2 promoters, respectively. In this study, we analyzed the differential expression of these promoters by using transcriptional fusion with the xylE reporter gene to study the activation of the sar locus. The data confirm the existence of three independent promoters with different promoter activities. Maximal promoter activity was observed with the combined fusion of P2-P3-P1 promoters. Expression studies with a sigB mutant revealed that the P3 promoter is SigB dependent. Analysis of these transcriptional fusions in a sarA mutant and in complemented strains with each of the sar transcriptional units revealed that the sar locus is autoregulatory, with SarA acting as a positive regulator. From various transcriptional fusion studies of the upstream region of the P1 promoter, we have localized a 34-bp sequence which seems to play a role in down-modulating P1 transcription. Using heparin-Sepharose and DNA-specific columns, we partially purified a 12-kDa protein, possibly a repressor, which binds to the promoter regions upstream of P2 and P1 and which also binds to the 34-bp sequence. These data indicated that the regulation of the sar locus is complex and may involve the sar gene product(s) and other regulatory protein(s).