Ribonuclease H (RNase H) belongs to the nucleotidyl-transferase (NT) superfamily and hydrolyzes the phosphodiester linkages that form the backbone of the RNA strand in RNA-DNA hybrids. This enzyme is implicated in replication initiation and DNA topology restoration and represents a very promising target for anti-HIV drug design. Structural information has been provided by high-resolution crystal structures of the complex RNase H/RNA center dot DNA from Bacillus halodurans (Bh), which reveals that two metal ions are required for formation of a catalytic active complex. Here, we use classical force field-based and quantum mechanics/molecular mechanics calculations for modeling the nucleotidyl transfer reaction in RNase H, clarifying the role of the metal ions and the nature of the nucleophile (water versus hydroxide ion). During the catalysis, the two metal ions act cooperatively, facilitating nucleophile formation and stabilizing both transition state and leaving group. Importantly, the two Mg2+ metals also support the formation of a metastable phosphorane intermediate along the reaction, which resembles the phosphorane intermediate structure obtained only in the debated P-phosphoglucomutase crystal (Lahiri, S. D.; et al. Science 2003, 299 (5615), 2067-2071). The nucleophile formation (i.e., water deprotonation) can be achieved in situ, after migration of one proton from the water to the scissile phosphate in the transition state. This proton transfer is actually mediated by solvation water molecules. Due to the highly conserved nature of the enzymatic bimetal motif, these results might also be relevant for structurally similar enzymes belonging to the NT superfamily.