Previous studies have shown that production of extracellular proteases in Staphylococcus aureus is stimulated by agr (RNAIII) and mgrA, and repressed by sarA. Protease expression is also repressed by rot, however this effect is generally observed only in agr mutants. Several other regulators (sarR, sarV, sarS, sae) that may impact protease expression have been described. As the interactions between all regulators that control protease gene expression are not fully understood, the present study was undertaken to elucidate the regulatory network governing aureolysin (aur) and staphylococcal serine protease (sspA) transcription. The regulation of both genes was studied as activation of the serine protease (SspA) zymogen requires aureolysin. For this purpose we have analyzed the effect of different combinations of regulatory mutations. The present study clearly shows that the positive effect of agr (RNAIII) on aur and sspA transcription requires rot, which is in accordance with the hypothesis that RNAIII acts by neutralizing Rot activity through binding [McNamara, P.J., Milligan-Monroe, K.C., Khalili, S., Proctor, R.A., 2000. Identification, cloning, and initial characterization of rot, a locus encoding a regulator of virulence factor expression in Staphylococcus aureus. J. Bacteriol. 182, 3197-3203]. Concomitantly, overexpression of rot in agr(+) strains or inactivation of rot in strains with low levels of RNAIII clearly affected aur and sspA transcription, indicating that the inhibiting effect of RNAIII on Rot could be titrated. Furthermore, our present data support that the only role of RNAIII in aur and sspA regulation is to counteract the repressive activity of Rot. Apart from an apparent direct positive effect of mgrA on sspA and aur transcription, these genes were mainly controlled through repression by sarA and rot, which seemed to occur via binding of SarA and Rot to the aur and sspA promoters, respectively. Maximum transcription of aur and sspA was obtained when both repressors were absent, either in a sarA mutant where Rot is neutralized by RNAIII during post-exponential phase, or in an agr sarA rot triple mutant. Interestingly, aur was much more sensitive to repression by sarA than by rot, whereas sspA was equally suppressed by sarA and rot. On the other hand, sspA was more sensitive to repression by rot than aur. Thus, SarA and Rot seemed to act independently in an additive way. Inactivation of sarR and sarS had no apparent effect on aur and sspA transcription, although overexpression of these regulators suppressed aur and sspA transcription, respectively, likely in a direct way as indicated by DNA binding experiments. In conclusion, our results indicate that aur and sspA transcription are coordinately regulated but can also be individually modulated by agr, sarA, rot, sarS, sarR, and mgrA. A provisional model for the regulation of aur and sspA transcription is presented.