The effects of protein synthesis inhibitors on the extent of O-acetylation of Neisseria gonorrhoeae peptidoglycan (PG) and on the resistance of PG to degradation by human PG hydrolases were examined. Addition of chloramphenicol, tetracycline, and streptomycin (in amounts equal to approximately twice their respective MICs) rapidly increased the level of O-acetylation of [3H]glucosamine-labeled N. gonorrhoeae FA19 PG from 46% to about 70% and simultaneously enhanced the resistance of the PG to degradation by human polymorphonuclear leukocyte lysozyme. Entry into the stationary phase also enhanced O-acetylation of FA19 PG, but neither protein synthesis inhibitors nor the stationary phase had a detectable effect on the O-acetyl-deficient, lysozyme-sensitive PG of N. gonorrhoeae RD5. Mild alkali treatment of PG derived from chloramphenicol-treated FA19 specifically removed O-acetyl groups and simultaneously reduced the extents of O-acetylation and polymorphonuclear leukocyte lysozyme resistance to the level of RD5 PG, suggesting that the O-acetyl substituents were solely responsible for the increased PG hydrolase resistance of PG from chloramphenicol-treated FA19. Pulse-chase experiments indicated that the drug-mediated enhancement of O-acetylation was limited to newly assembled PG. In summary, conditions favoring unbalanced macromolecular synthesis and bacteriostasis increased the level of O-acetylation and the PG hydrolase resistance of gonococcal PG. Similar conditions encountered by gonococci in vivo might potentiate the pathobiological consequences of PG-host interactions.