An individual strain of Neisseria gonorrhoeae may produce up to 16 different DNA methytransferases (MTases). We have used a novel cloning system that is able to detect MTase clones in the absence of direct selection [Piekarowicz et al., Nucleic Acids Res. 19 (1991) 1831-1835] to identify 14 different MTase clones. Initial characterization of these clones indicates that at least seven of these MTases are linked to restriction endonuclease (ENase) systems. Six of these systems have been characterized by DNA sequence analysis, and the open reading frames encoding each of these systems have been identified. The recognition sequences for the cloned systems have the following specificities: S.NgoI, RGCGCY; S.NgoII, GGCC; S.NgoIV, GCCGCC; S.NgoV, GGNNCC; S.NgoVII, GCSGC; S.NgoVIIIA, GGTGA; and S.NgoVIIIC, TCACC. Of those systems that have been cloned, NgoI-NgoVII are typical type II R-M systems, with each encoding a DNA MTase that methylates cytosine in position 5. NgoVIII is a type IIS system, containing an ENase and two different MTases. One of these is a cytosine MTase (NgoVIIIC) and the other is an adenine MTase (NgoVIIIA). Although most of our clones encodes both the ENase and the MTase, none of the six R-M systems are genetically linked on the chromosome.