RNase H is a prototypical example for two-metalion catalysis in enzymes. An RNase H activity cleaving the ribonucleic acid (RNA) backbone of a DNA/RNA hybrid is present not only in important drug targets, such as the HIV-1 reverse transcriptase, but also in many other nudeases, such as Homo sapiens (Hs) and Escherichia coil (Ec) RNase H or, notably, in enzymes that are part of the CRISPR gene editing molecular machinery. Despite its importance, the reaction mechanism uncovering the proton-transfer events is not yet understood. In particular, it is not known, which group is the proton donor for the leaving group. Moreover, several different proton acceptors were proposed, and the exact identity of the proton acceptor is also elusive. Here, we revisit the mechanism for RNAse H, whereby we find that the highly conserved Glu residue of the DDE motif acts as a proton donor via a mechanism further stabilized by the 2'O atom of the sugar. Additionally, we also describe an alternative proton-transfer mechanism via a consented catalytic His residue to deprotonate the attacking water molecule. Furthermore, our quantum mechanics/molecular mechanics (QM/MM) calculations combining Hamiltonian replica exchange with a finite-temperature string method provide an accurate free-energy profile for the reaction catalyzed by the HIV-1 RNase H. Our reported pathway is consistent with kinetic data obtained for mutant HIV-1, Hs, and Ec RNase H, with the calculated pK(a), values of the DEDD residues and with crystallographic studies. The overall reaction barrier of similar to 19 kcal mol(-1), encountered in the phosphate-cleavage step, matches the slow experimental rate of similar to 1-100 min(-1) Additionally, using molecular dynamics (MD) calculations, we sample the recently identified binding site for a third transient divalent metal ion in the vicinity of the scissile phosphate in the product complex. Our results account for the experimental observation of a third metal ion facilitating product release in an Aquifex aeolicus RNase III crystal structure and the Bh RNase H in crystallo reaction. Taken together, we provide a molecular mechanism of the nuclease catalytic reaction that is likely common for the broad family of twometal-ion catalytic phosphate-cleaving enzymes with a DDE motif.