Biochemical properties of Type II restriction enzyme BanI were characterized, Kinetic parameters were evaluated and an enhancement of rate was observed when the recognition site was located in a more central position in the substrate, suggesting that BanI locates its recognition site by a sliding mechanism, As BanI has three cysteine residues in its primary sequence, the effect of thiol inhibitors on BanI activity was also studied. Partial inhibition was observed only at a very high concentration of the inhibitor indicating that cysteine residues are not directly involved in catalysis. The gel electrophoretic mobility shift assay demonstrated specific complex formation between BanI and the DNA substrate in the presence of poly dI-dC and Mg2+. A secondary structure analysis and comparison with EcoRI and BamHI crystal structure revealed a putative active site similar to that seen in BamHI but different in the order in which the catalytic domain (central beta -sheet) and recognition domain (adjacent alpha -helix) were arranged in the protein,