The methyltransferase M1.NcuI is a member of the restriction-modification system in Neisseria cuniculi ATCC14688 and recognizes the asymmetric pentanucleotide sequence 5'-GAAGA-3'/3'-CTTCT-5'. We purified M1.NcuI to electrophoretic homogeneity using a four-step chromatographic procedure. M1.NcuI is a protein with M(r)=32,000+/-1000 under denaturing conditions. It modifies the recognition sequence by transferring the methyl group from S-adenosyl-l-methionine to the 3' adenine of the pentanucleotide sequence 5'-GAAGA-3'. M1.NcuI, like many other methyltransferases, occurs as a monomer in solution, as determined by gel filtration. Divalent cations inhibit the methylation activity of M1.NcuI. Optimal enzyme activity was observed at a pH of 8.0. M1.NcuI cross-reacted with anti-M1.MboII serum which reflects the similarity of M1.NcuI with M1.MboII at the amino acid level. The gene coding for the enzyme, designated ncuIM1, was cloned, sequenced and overexpressed in Escherichia coli. The structural gene is 780 nucleotides in length coding for a protein of 259 amino acids (M(r) 30,098). The presence and distribution of nine highly conserved amino acid sequence motifs and a putative target recognition domain in the enzyme structure suggest that M1.NcuI, similar to M1.MboII and M1.HpyAII, belongs to N(6)-adenine beta-class DNA methyltransferases.