We have used the intrinsic tryptophan fluorescence of the EcoRV restriction endonuclease to monitor changes in protein conformation during binding and cleavage of a duplex oligodeoxynucleotide substrate. Appropriate conditions for single-turnover reactions were first determined by steady-state kinetics. When single turnovers were monitored by stopped-flow fluorescence, the mixing together of EcoRV, oligonucleotide and MgCl2 resulted in a rapid increase in tryptophan fluorescence followed by a slow decrease. Further analysis by order-of-mixing and quench experiments showed that the transient increase in fluorescence was due to a conformational change coupled to DNA binding, while the subsequent decay was concomitant with phosphodiester hydrolysis. The rate of the latter step varied with the concentration of Mg2+ ions, but another Mg(2+)-dependent transition was observed upon the addition of MgCl2 to a preformed enzyme-DNA complex. These results lead to a reaction scheme in which one Mg2+ binds to the active site prior to phosphodiester hydrolysis but a second Mg2+ is then needed to carry out the hydrolytic reaction. This scheme is correlated to the crystal structures of the EcoRV endonuclease and its complexes with DNA and Mg2+ ions.