The Cfr9I restriction endonuclease recognizes and cleaves duplex DNA sequence C decreases CCGGG. The binding of restriction endonuclease Cfr9I to DNA was examined in the absence of Mg2+ using gel-mobility-shift and nitrocellulose-filter-binding assays. It was shown that restriction endonuclease Cfr9I bound DNA fragments either containing or lacking the canonical recognition sequence with equal affinity. These results suggest that the specificity of restriction endonuclease Cfr9I is expressed during the catalytic step. The cleavage of supercoiled pUC18 DNA by restriction endonuclease Cfr9I showed that at low concentrations of MgCl2, only with open-circular DNA, nicks appeared in one strand at the recognition sequence, while the cleavage of the second strand was very slow. At higher concentrations of MgCl2 the enzyme cleaves either one or both strands of the DNA. Under these conditions the supercoiled DNA was converted to open-circular and linear forms simultaneously rather than consecutively. It was shown that open-circular DNA was a poor substrate for restriction endonuclease Cfr9I. These results suggested that both Mg2+ and intact recognition sequence are required to drive the enzyme into correct conformation to ensure DNA cleavage.