Ca(2+)-binding proteins are widely distributed throughout cells and play various important roles. Calbindin D9k is a member of the EF-hand Ca(2+)-binding protein family. In this study, we examined the binding of Ca(2+) to calbindin D9k in terms of the free energy of solvation, as obtained by 3D reference interaction site model theory, which describes the statistical mechanics of liquids. We also investigated the main structural biological factor using spatial decomposition analysis in which the solvation free energy values are decomposed into the residue. We found some characteristic residues that contribute to stabilization of the holo-structure (Ca(2+)-binding structure). These results indicated that, in the holo-structure, these residues are newly exposed to solvent. Subsequently, the gain in solvation free energy, involving a conformational change and exposure to solvent, forms the driving force for binding of the Ca(2+) ion to the EF-hand.