A 350 base pair Neisseria gonorrhoeae DNA restriction fragment was cloned after subtractive hybridization to Neisseria meningitidis DNA. This restriction fragment hybridized to 105 out of 106 N. gonorrhoeae strains tested. While three N. meningitidis strains did not hybridize to this probe, Neisseria mucosa DNA exhibited cross-hybridization. This particular clone was used to screen a N. gonorrhoeae genomic DNA library. A positive 2.4 kilobase pair clone was shown by DNA sequencing to contain two long open reading frames. One open reading frame did not hybridize to N. mucosa and other Neisseria species, while it retained specificity for the original 105 N gonorrhoeae strains. This open reading frame also showed significant homology to cytosine DNA methyltransferases.