Neisseria gonorrhoeae biofilms have been shown to contain lipooligosaccharide and single-stranded DNA, both of which could be released by growing bacteria. However, they also contain substantial amounts of double-stranded DNA, which is presumably released by bacterial cell lysis. Thus, we investigated whether cell wall peptidoglycan might be released by autolysis and contribute to biofilms and whether the peptidoglycan structure could affect biofilm amounts. We used strains non-variable for pilin sequence and lacking opacity protein genes to ensure that spontaneous changes in these factors would not occur and affect aggregation and adhesion. Gonococci grown in 24-well plates showed substantial biofilm formation, and pacA mutants lacking O-acetylation of the peptidoglycan showed increased biofilm amounts. Addition of magnesium did not diminish the increased biofilm of a pacA mutant, indicating that membrane instability was not the cause of the increased biofilm. We used mutanolysin to degrade extracellular peptidoglycan and found that the bacteria formed no biofilm at all in the presence of the enzyme, whereas boiled mutanolysin had no effect. Ongoing experiments are examining the effects of lysozyme on biofilm formation and differences in biofilm amounts affected by peptidoglycan peptide structure. Although gonococci are unusual among biofilm producing species in not making an exopolysaccharide, they use internal polysaccharides DNA and peptidoglycan as essential material to form the biofilm.