The peptidoglycan compositions of vancomycin-resistant Staphylococcus aureus (VRSA) strain Mu50 (MIC 8 mg/L) and hetero-VRSA strain Mu3 (MIC 3 mg/L) were compared in order to understand the mechanism of vancomycin resistance. As compared with Mu3, the cell wall of Mu50 had increased amounts of glutamine-non-amidated muropeptides and decreased cross-linking of peptidoglycan with a greatly decreased dimer/monomer ratio of muropeptides. In agreement with this observation, the peptidoglycan of Mu50 bound 1.4 times more vancomycin than that of Mu3. The increase in non-amidated muropeptides and the reduced cross-linking of the cell-wall peptidoglycan may contribute to the vancomycin resistance by increasing the consumption of vancomycin by the pre-existing cell wall of Mu50 and reducing the amount of vancomycin reaching the cytoplasmic membrane where the vital targets of the antibiotic are situated.