The temporal expression of most virulence factors in Staphylococcus aureus is regulated by pleiotropic loci such as agr and sar. We have previously shown that the sar locus affects hemolysin production because it is required for agr transcription. To delineate the sar genetic determinant required for agr transcription, single copies of fragments from the sar locus, encompassing the individual sar transcripts (sarA, sarC, and sarB), were introduced into a sar mutant via the integration vector pCL84. Although a DNA fragment encompassing the sarA transcript plus a 189-bp upstream region was sufficient for agr expression, complementation analysis revealed that the sarB transcript was the most effective in augmenting agr transcription as determined by RNAII and RNAIII transcription and gel retardation assays with the P2 and P3 promoters of agr. As the region upstream of the sarA transcript encodes a 39-amino-acid open reading frame, ORF3, it is possible that posttranslational cooperation between the sarA gene product and ORF3 may be necessary for optimal agr expression. Deletion studies demonstrated that an intact sarA gene is essential for agr transcription. However, mutagenesis and in vitro translation studies revealed that unlike the agr locus, the required element is the SarA protein and not the RNA molecule. Taken together, these results indicate that the sarA-encoded protein, possibly in conjunction with peptides encoded in the upstream region, regulates hemolysin production by controlling agr P2 and P3 transcription.