Abstract The structure of peptidoglycan extracted from the Gram-positive bacterium Sarcina ventriculi grown at pH 3 was characterized by amino acid analysis, mass spectrometry, and two-dimensional NMR spectroscopy. The basic muropeptide subunit consisted of an N-acetylglucosamine- β-1,4- N-acetylmuramic acid disaccharide substituted with an oligopeptide with the sequence Ala- isoGln-A 2pm(-Gly)-Ala. The dimeric muropeptide was also characterized as a cross-linked bis-disaccharide-penta-hexapeptide with the structure, GlcNAc-MurNAc-Ala- isoGln-A 2pm(-Gly)-Ala → GlcNAc-MurNAc-Ala- isoGln-A 2(-Gly)-Ala-Ala. These results are consistent with a structure proposed based on enzymatic degradation and chemical modifications but with no use of spectroscopic information [O. Kandler, D. Claus, and A. Moore, Arch. Mikrobiol., 82 (1972) 140–146]. The cell wall of this organism is very tightly cross-linked and is much more rigid than that of most other Gram-positive bacteria. There is, however, a large degree of conservation in the general structure compared to peptidoglycan in other bacteria that are not well adaptable to extremes, indicating that the membrane plays a more important role in adaptation.