We have cloned from Neisseria gonorrhoeae MS11 the gene encoding a methylase that modifies the sequence GCCGGC. The corresponding restriction enzyme was also encoded by this clone. Sequence analysis demonstrated that the methylase shares sequence similarities with other cytosine methylases, but the sequence organization of M.NgoMI is different from that seen for other cytosine methylases. A deletion was introduced into the chromosome of N. gonorrhoeae MS11 to produce strain MUG701, a strain that is inactivated in both the methylase and the restriction genes. Although this strain no longer methylated its DNA at the NgoMI recognition sequence, cells were viable and had no other significant phenotypic changes. Transformation data indicated that MS11 does not produce enough restriction activity to block plasmid transformation in the gonococcus, even though restriction activity could be demonstrated in E. coli containing the cloned gene.