Low affinity of penicillin for altered penicillin-binding protein 2 (PBP 2) of Neisseria gonorrhoeae that are chromosomally resistant to penicillin (CMRNG) has been attributed, at least partly to the insertion of an additional aspartic acid (Asp-35A) codon in the penA gene. Oligonucleotide probes were used to detect the presence or absence of this extra amino-acid residue in 34 non-penicillinase producing N. gonorrhoeae with decreased susceptibility to penicillin (MICs 0.25-2 mg/L) and 11 strains sensitive to penicillin (MIC < or = 0.06 mg/L) isolated in Greece and previously characterized by auxotype and serovar. The Asp-345A codon was found in all CMRNG strains as well as in strains with intermediate resistance to penicillin, but was absent from all strains exhibiting an MIC of penicillin of < or = 0.03 mg/L. No association of the Asp-345A insertion to any particular auxotype or serovar was found. These results expand the previously reported correlation between the presence of the Asp-345A codon and reduced sensitivity to penicillin, and shows that this particular mutation is common among epidemiologically distinct CMRNG strains.