The staphylococcal accessory regulator (encoded by sarA) is an important global regulator of virulence factor biosynthesis in Staphylococcus aureus. To further characterize its role in virulence determinant production, an sarA knockout mutant was created by insertion of a kanamycin antibiotic resistance cassette into the sarA gene. N-terminal sequencing of exoproteins down-regulated by sarA identified several putative proteases, including a V8 serine protease and a novel metalloprotease, as the major extracellular proteins repressed by sarA. In kinetic studies, the sarA mutation delays the onset of alpha-hemolysin (encoded by hla) expression and reduces levels of hla to approximately 40% of the parent strain level. Furthermore, SarA plays a role in signal transduction in response to microaerobic growth since levels of hla were much lower in a microaerobic environment than after aerobic growth in the sarA mutant. An exoprotein exhibiting hemolysin activity on sheep blood, and up-regulated by sarA independently of the accessory gene regulator (encoded by agr), was specifically induced microaerobically. Transcriptional gene fusion and Western analysis revealed that sarA up-regulates both toxic shock syndrome toxin 1 gene (tst) expression and staphylococcal enterotoxin B production, respectively. This study demonstrates the role of sarA as a signal transduction regulatory component in response to aeration stimuli and suggests that sarA functions as a major repressor of protease activity. The possible role of proteases as regulators of virulence determinant stability is discussed.