Staphylococcus aureus is a medically important pathogen that synthesizes a wide range of virulence determinants. The synthesis of many staphylococcal virulence determinants is regulated in part by stress-induced changes in the activity of the tricarboxylic acid (TCA) cycle. One metabolic change associated with TCA cycle stress is an increased concentration of ribose, leading us to hypothesize that a pentose phosphate pathway (PPP)-responsive regulator mediates some of the TCA cycle-dependent regulatory effects. Using bioinformatics, we identified three potential ribose-responsive regulators that belong to the RpiR family of transcriptional regulators. To determine whether these RpiR homologues affect PPP activity and virulence determinant synthesis, the rpiR homologues were inactivated, and the effects on PPP activity and virulence factor synthesis were assessed. Two of the three homologues (RpiRB and RpiRC) positively influence the transcription of the PPP genes rpiA and zwf, while the third homologue (RpiRA) is slightly antagonistic to the other homologues. In addition, inactivation of RpiRC altered the temporal transcription of RNAIII, the effector molecule of the agr quorum-sensing system. These data confirm the close linkage of central metabolism and virulence determinant synthesis, and they establish a metabolic override for quorum-sensing-dependent regulation of RNAIII transcription.