The mechanism of action of the EcoRV restriction endonuclease at its single recognition site on the plasmid pAT153 was analyzed by kinetic methods. In reactions at pH 7.5, close to the optimum for this enzyme, both strands of the DNA were cut in a single concerted reaction: DNA cut in only one strand of the duplex was neither liberated from the enzyme during the catalytic turnover nor accumulated as a steady-state intermediate. In contrast, reactions at pH 6.0 involved the sequential cutting of the two strands of the DNA. Under these conditions, DNA cut in a single strand was an obligatory intermediate in the reaction pathway and a fraction of the nicked DNA dissociated from the enzyme during the turnover. The different reaction profiles are shown to be consistent with a single mechanism in which the kinetic activity of each subunit of the dimeric protein is governed by its affinity for Mg2+ ions. At pH 7.5, Mg2+ is bound to both subunits of the dimer for virtually the complete period of the catalytic turnover, while at pH 6.0 Mg2+ is bound transiently to one subunit at a time. The kinetics of the EcoRV nuclease were unaffected by DNA supercoiling.