We found an increased abundance of pbpB-specific transcripts in vancomycin intermediate-resistant Staphylococcus aureus (VISA) isolates compared with that found in paired, genetically identical, susceptible isolates. This difference in expression cannot be explained by differences in the pbpB promoter sequence. Since the factors controlling pbpB gene expression have remained largely unexplored, various conditions that might affect pbpB transcript abundance were examined. In both vancomycin-susceptible and VISA strains, pbpB expression varied with the growth phase, with the highest abundance of pbpB-specific transcripts detected during mid-log phase. Interestingly, both vancomycin and oxacillin were able to induce pbpB transcription above a constitutive level. When vancomycin was absent, one of the three pbpB-specific transcripts that were usually faintly detected in non-VISA strains was more readily detected in VISA strains during mid-log but not stationary phase. This transcript was enhanced in non-VISA strains by vancomycin induction. Gel shift assays indicated that an increased amount of the putative transcription factor that binds to both P1 and P1' promoter regions is present in the cytosol of vancomycin-induced cells. Neither the SigB sigma factor nor the quorum-sensing agr locus was required for growth phase-variable pbpB expression or transcriptional induction of pbpB by vancomycin or oxacillin. Also, MecI, MecR1, BlaI, and BlaR1, regulatory proteins that mediate beta-lactam-inducible expression of mecA and beta-lactamase, were not required for antibiotic induction of pbpB transcription. These data support the idea that pbpB expression is modulated by a trans-acting factor in response to the presence of the cell wall-active antibiotics vancomycin and oxacillin.