The composition and structure of peptidoglycan (murein) extracted from the extreme thermophilic eubacterium Thermus thermophilus HB8 are presented. The structure of 29 muropeptides, accounting for more than 85% of total murein, is reported. The basic monomeric subunit consists of N-acetylglucosamine-N-acetylmuramic acid-L-Ala-D-Glu-L-Orn-D-Ala-D-Ala, acylated at the delta-NH2 group of Orn by a Gly-Gly dipeptide. In a significant proportion (about 23%) of total muropeptides, the N-terminal Gly is substituted by a residue of phenylacetic acid. This is the first time phenylacetic acid is described as a component of bacterial murein. Possible implications for murein physiology and biosynthesis are discussed. Murein cross-linking is mediated by D-Ala-Gly-Gly peptide cross-bridges. Glycan chains are apparently terminated by (1-->6) anhydro N-acetylmuramic acid residues. Neither reducing sugars nor murein-bound macromolecules were detected. Murein from T. thermophilus presents an intermediate complexity between those of gram-positive and gram-negative organisms. The murein composition and peptide cross-bridges of T. thermophilus are typical for a gram-positive bacterium. However, the murein content, degree of cross-linkage, and glycan chain length for T. thermophilus are closer to those for gram-negative organisms and could explain the gram-negative character of Thermus spp.