The fine structure of sacculi from Thermus thermophilus HB27, T. aquaticus YT-1 and Thermus ATCC27737 has been worked out by HPLC analysis and mass spectrometry techniques. The three microorganisms have a murein composition of the rare A3beta chemotype, but showed substantial differences in muropeptide composition. Phenylacetylated muropeptides,previously described in T. thermophilus HB8, were detected exclusively in T. thermophilus HB27. Murein from T. aquaticusYT-1 was devoid of D-Ala-D-Ala terminated muropeptides, which were, in contrast, abundant in T. thermophilus HB27 and Thermus ATCC27737. The significance of these findings is discussed.