The reaction mechanism of phosphate monoester hydrolysis in alkaline phosphatase is analyzed by means of hybrid QM/MM simulations. A recently developed semiempirical Hamiltonian, AMl/d-PhoT, which takes into account the d orbitals on the phosphorus atom, has been employed. The reaction mechanism obtained is either associative or dissociative, depending on the size of the QM subsystem. The results are rationalized on the basis of the degree of charge transfer from the reacting fragments to the two zinc ions present in the active site, which has been observed to be dependent on whether or not metal atoms and their coordination spheres are included in the QM region. The description obtained using the largest QM region agrees with the picture obtained from experimental data. © 2009 American Chemical Society.