We here present a theoretical study of the alkaline hydrolysis of a phosphodiester (methyl p-nitrophenyl phosphate or MpNPP) in the active site of Escherichia coli alkaline phosphatase (AP), a monoesterase that also presents promiscuous activity as a diesterase. The analysis of our simulations, carried out by means of molecular dynamics (MD) simulations with hybrid quantum mechanics/molecular mechanics (QM/MM) potentials, shows that the reaction takes place through a D NA N or dissociative mechanism, the same mechanism employed by AP in the hydrolysis of monoesters. The promiscuous activity observed in this superfamily can be then explained on the basis of a conserved reaction mechanism. According to our simulations the specialization in the hydrolysis of phosphomonoesters or phosphodiesters, developed in different members of the superfamily, is a consequence of the interactions established between the protein and the oxygen atoms of the phosphate group and, in particular, with the oxygen atom that bears the additional alkyl group when the substrate is a diester. A water molecule, belonging to the coordination shell of the Mg 2+ ion, and residue Lys328 seem to play decisive roles stabilizing a phosphomonoester substrate, but the latter contributes to increase the energy barrier for the hydrolysis of phosphodiesters. Then, mutations affecting the nature or positioning of Lys328 lead to an increased diesterase activity in AP. Finally, the capacity of this enzymatic family to catalyze the reaction of phosphoesters having different leaving groups, or substrate promiscuity, is explained by the ability of the enzyme to stabilize different charge distributions in the leaving group using different interactions involving either one of the zinc centers or residues placed on the outer side of the catalytic site. © 2011 American Chemical Society.