The control of virulence determinant expression in Staphylococcus aureus is a complex process involving global regulatory loci such as sar and agr. The sar locus consists of a 372 bp sarA open reading frame (ORF) preceded by a triple promoter region interspersed with two putative smaller ORFs (ORF3 and ORF4). The triple promoter system yields three overlapping sar transcripts (sarA, sarC and sarB of 0.56, 0.8 and 1.2 kb respectively). We have recently shown that the SarA protein binds to the agr promoter region to stimulate the transcription of RNAII and RNAIII, two major transcripts encoded within the agr locus. To assess the role of the region upstream of sarA in agr expression, we evaluated the contribution of ORF3 and ORF4 to SarA protein expression and to agr activation by introducing nonsense mutations into the respective ORFs. Northern analysis of sar mutant clones containing these mutations carried on a shuttle plasmid revealed that all three sar-related transcripts are present. Using anti-SarA monoclonal antibodies with defined epitopes in a competitive ELISA to determine the SarA protein level, we found that the introduction of a stop codon in ORF3 on a shuttle plasmid carrying the intact sar locus in a sar mutant led to a significant decrease in SarA protein level compared with the non-mutated control. The effect of a nonsense mutation in ORF4 on SarA levels is much less. Likewise, an analogous sar mutant clone with a deletion in ORF3 also displayed a lower SarA level than its intact counterpart. The reduction in SarA expression correlated with a lower level of agr activation in the corresponding sar mutant clone. These data suggest that ORF3, and to a lesser degree ORF4, may affect agr expression by modulating SarA protein expression.