The extent of lysozyme resistance and O-acetylation of purified peptidoglycan (PG) from 20 strains of Neisseria gonorrhoeae was examined to determine how widespread these properties are among various subsets of gonococcal isolates. To determine digestibility by lysozyme, we treated [3H]- or [14C]glucosamine-labeled PG with hen egg white lysozyme (HEW-LZ) and determined the size distribution of HEW-LZ soluble PG at the completion of the reaction by molecular-sieve high-performance liquid chromatography, using a Varian TSK SW2000 column, a method that proved considerably more efficient than traditional chromatography for fractionating low-molecular-weight PG fragments solely on the basis of size. The extent of HEW-LZ resistance was expressed as the percentage of PG that was larger in size than disaccharide peptide tetramers (including insoluble PG removed by centrifugation). The percent O-acetylation was determined by converting insoluble PG totally to uncross-linked monomers by the combined action of Chalaropsis B muramidase followed by Escherichia coli endopeptidase and then quantitating radioactivity in O-acetylated and non-O-acetylated monomers after paper chromatography. The PG of the vast majority (19 of 20) of gonococcal strains examined was extensively HEW-LZ resistant (range, 40 to 60% larger than tetramers) and extensively O-acetylated (range, 34 to 52%). Only the PG of strain RD5 (highest rate of PG turnover among gonococci so far examined and the prototype of gonococci having O-acetyl-deficient PG) had greatly reduced O-acetylation (15%) and exhibited virtually no HEW-LZ resistance (2% larger than tetramers). Extensive HEW-LZ resistance and O-acetylation were apparently not associated specifically with (i) a given type of colonial variant (piliated versus nonpiliated or opaque versus transparent), (ii) a given type of clinical isolate (local versus disseminated), (iii) the extent of laboratory passage, or (iv) (with the possible exception of penicillin-resistant strain FA102) the presence of one or more genetic loci governing antibiotic resistance among members of an isogenic set of gonococci. From this survey, we conclude that lysozyme resistance and extensive O-acetylation of PG are widespread among gonococci and, thus, that most strains are potential sources of hydrolase-resistant PG that conceivably could persist as macromolecular fragments in vivo.