We previously reported the first vancomycin-resistant Staphylococcus aureus (VRSA) clinical strain, Mu50, whose cell wall is remarkably thickened resulting from the activation of cell-wall synthesis. To explore the genetic basis for the vancomycin resistance, cDNA differential hybridization was performed using: RNAs extracted from a set of closely related S. aureus strains with various levels of vancomycin susceptibilities. The strains were Mu3 (MIC = 2 mu g/ml), Mu50 (MIC = 8 mu g/ml), and a susceptible revertant of Mu50, Mu50w (MIC = 0.5 mu g/ml). in this study, we report identification of a novel response regulator, designated vraR (standing for vancomycin-resistance associated gene R) whose transcription was remarkably up-regulated in Mu3 and Mu50 as compared to Mu50w. Experimental over-expression of VraR in vancomycin-susceptible strain N315P raised vancomycin resistance of the strain. Also, the genes coding for fructose utilization, fatty acid metabolism and two putative ATP-binding cassette (ABC) transporter genes were found to be up-regulated in Mu3 and Mu50. On the other hand, Protein A expression was suppressed in Mu50, as compared with Mu3 and Mu50w. We consider that the response regulator vraR is one of the key regulators modulating the level of vancomycin-resistance in S. aureus. Presumed increased uptake of fructose and altered fatty acid metabolism may also contribute to vancomycin resistance by supplying more precursor metabolites for cell-wall synthesis.