The Type IIS restriction endonuclease MnlI recognizes the non-palindromic nucleotide sequence 5V-CCTC(N)7/6, and cleaves DNA strands as indicated by the arrow. The genes encoding MnlI restriction -modification system were cloned and sequenced. It comprises N6methyladenine and C5-methylcytosine methyltransferases and the restriction endonuclease. Biochemical studies revealed that MnlI restriction endonuclease cleaves double-and single-stranded DNA, and that it prefers different metal ions for hydrolysis of these substrates. Mg 2+ ions were shown to be required for the specific cleavage of double-stranded DNA, whereas Ni 2+ and some other transition metal ions were preferred for nonspecific cleavage of single-stranded DNA. The C-terminal part of MnlI restriction endonuclease revealed an intriguing similarity with the H-N-H type nucleolytic domain of bacterial toxins, Colicin E7 and Colicin E9. Alanine replacements in the conserved sequence motif 306Rx 3 ExHHx 14 Nx 8 H greatly reduced specific activity of MnlI, and some mutations even completely inactivated the enzyme. However, none of these mutations had effect on MnlI binding to the specific DNA, and on its oligomerisation state as well. We interpret the presented experimental evidence as a suggestion that the motif 306Rx 3 ExHHx 14 Nx 8 H represents the active site of MnlI. Consequentially, MnlI seems to be the member of Type IIS with the active site of the H-N-H type.