Whole cells and cell-wall fractions of Staphylococcusaureus have been labeled by various combinations of [1-13C]glycine, [15N]glycine, L--6-13C-lysine, L--6-15N-lysine, D--1-13C-alanine, and D--15N-alanine. The resulting materials have been examined using 13C and 15N solid-state, magic-angle spinning NMR techniques including cross-polarization, double cross-polarization, and rotational-echo double resonance. The results of these measurements indicate that the peptidoglycan glycyl bridges are complete (five units long) and form cross-links between three-quarters of all peptide stems. The pentaglycyl bridges are immobilized in lyophilized cell-wall fractions in a compact conformation with inter-residue spacings comparable to those of an alpha helix. The bridges have a similar compact conformation in intact whole cells, regardless of whether the cells have been lyophilized or were hydrated and frozen at -10 degrees C. The bridges are also in a time-averaged compact conformation in whole cells at 0 degrees C but with sizable structural fluctuations associated with local mobility. A small fraction of bridges are in extended-chain conformations.