The roles of divalent metal ions in DNA cleavage by the EcoRV endonuclease were studied by using Co 2 + or Mn 2 + as substitutes for the natural cofactor Mg 2 + . In steady-state experiments with a 12 bp oligonucleotide substrate, Co 2 + yielded a similar turnover rate to that with Mg 2 + , but Mn 2 + gave a slower rate. Single turnovers of EcoRV on this substrate were analysed by stopped-flow and quench-flow methods, to determine the rates for the formation of the ternary enzyme-DNA-metal complex, the hydrolysis of the phosphodiester bonds and the dissociation of the cleaved DNA. With Co 2 + , all three steps had similar rates to those with Mg 2 + . In contrast, Mn 2 + gave a faster rate for phosphodiester hydrolysis than either Mg 2 + or Co 2 + , but a slower rate for product dissociation, thus accounting for its low turnover rate. Single turnovers on plasmids also yielded faster rates for substrate hydrolysis with Mn 2 + compared to Mg 2 + and Co 2 + . Since Mn 2 + gave the most rapid rates for the hydrolytic step, despite being less electronegative than Co 2 + , the function of the metal ion at the active site of EcoRV cannot be just the polarisation of the scissile phosphate. Moreover, the minimal scheme for the Co 2 + -catalysed reaction requires two metal ions for DNA cleavage. The metal ions seem to be involved in the precise positioning of both the substrate and the water that acts as the attacking nucleophile and in activating that water molecule. A model is presented to account for how two metal ions might fulfil these functions.