The role of Mg2+ ions during precursor formation in DNA hydrolysis by the homodimeric restriction enzyme EcoRV was elucidated based on the 3D-reference interaction site model (RISM) theory and the molecular dynamics (MD) simulation. From an analysis of the spatial distribution of Mg2+ in an active site using 3D-RISM, we identified a new position for Mg2+ in the X-ray EcoRV-DNA complex structure (1rvb). We refer to the position as site IV dagger. Site IV(dagger )is almost at the same position as that of a Ca2+ ion in the superimposed X-ray crystallographic active-site structure of the PvuII-DNA complex (1f0o). 3D-RISM was also used to locate the position of water molecules, including the water nucleophile at the active site. MD simulations were carried out with the initial structure having two Mg2+ ions at site IV dagger and at site I*, experimentally identified by Horton et al., to find a stable complex structure in which the DNA fragment was rearranged to orient the scissile bond direction toward the water nucleophile. The equilibrium active-site structure of the EcoRV-DNA complex obtained from the MD simulation was similar to the superimposed X-ray crystallographic structure of the BamHI-DNA complex (2bam). In the active-site structure, two metal ions have almost the same position (<1.0 angstrom) as that of 2bam, and the scissile phosphate is twisted to orient the scissile bond toward the water nucleophile, as is the case in 2bam. We propose the equilibrium active-site structure obtained in this study as a precursor for the hydrolysis reaction of EcoRV.