Cross-polarization magic-angle spinning and rotational-echo double resonance 13C and 15NNMR experiments have been performed on intact cells of Staphylococcus aureus labeled withd-[1-13C]alanine and [15N]glycine or with [1-13C]glycine and l-[ε-15N]lysine. The cells were harvestedduring stationary or exponential growth conditions, the latter in media with and without the addition ofvancomycin. The results of these experiments allowed the in situ determination of the relative concentrationsof peptidoglycan cross-links (the number of peptide-stem d-alanines covalently linked to a pentaglycylbridge) and bridge-links (the number of peptide-stem lysines covalently linked to a pentaglycyl bridge).The concentration of cross-links remained constant in the presence of vancomycin, whereas the numberof bridge-links decreased. These changes suggest that vancomycin (at therapeutic levels) interruptspeptidoglycan synthesis in S. aureus by interference with transglycosylation.