Muramidase digests of alkali-treated SDS-insoluble peptidoglycan from two strains of Neisseria gonorrhoeae were examined. Both strains contained disaccharide peptide monomers that had intramolecular 1,6-anhydro-muramyl ends. In contrast to strain 1L260, in which 50% of the monomer fraction is 0-acetylated, the monomer fraction from strain RD, was completely devoid of 0-acetyl groups, as shown by HPLC. Penicillin decreased the 0-acetylation of peptidoglycan but did not affect the proportion of anhydro-muramyl residues.