The effect of glucose on accessory gene regulator (agr) expression in Staphylococcus aureus was examined. agr is a global regulator that affects the expression of numerous genes, including those for some factors implicated in virulence, such as toxic shock syndrome toxin 1, alpha-hemolysin, and protein A. The agr locus determines two divergent transcripts, designated RNAII and RNAIII. RNAII contains four open reading frames (agrABCD), and RNAIII encodes delta-hemolysin. The mechanisms responsible for agr-mediated regulation are not well understood, but it appears that the RNAIII transcript plays a central role in the regulation of a number of target genes, including those for alpha-hemolysin (hla), beta-hemolysin (hlb), protein A (spa), and staphylococcal enterotoxin B (seb+). In this study, S. aureus cultures were grown either in a shake flask system with a complex medium or in a fermentor system with a completely defined medium in which the pH and glucose concentration were maintained. Northern (RNA) blot analysis revealed that a dramatic reduction in agr expression was apparent only when the cultures contained glucose and when the pH was 5.5 or was not maintained. The effect of glucose on two agr target genes, sec+ and hla, was also studied. Glucose-containing cultures produced less sec+ and hla mRNAs at maintained pH (6.5). In addition, the glucose effect on sec+ and hla was enhanced under conditions that inhibited agr expression (i.e., pH 5.5 or a nonmaintained pH).