The initial Mn2+ binding site in photoassembly of the Mn4CaO5 cluster, the catalytic center of water oxidation in photosystem II, was studied using quantum mechanics/molecular mechanics calculations. Among the five metal sites (Mn1-Mn4 and Ca) of the Mn4CaO5 cluster, the Mn1 site involving D1-H332 was most stable for Mn(2+ )binding, while the Mn2 site showed a slightly higher energy comparable to the thermal fluctuation energy at room temperature. It is thus suggested that Mn2+ binding at the Mn1 site, likely in equilibrium with that at the Mn2 site, is the initial step of photoassembly of the Mn4CaO5 cluster.