The thermodynamic change in the binding of Ca2+ to a mutant human lysozyme having an engineered Ca2+ binding site (Kuroki, R., Taniyama, Y., Seko, C., Nakamura, H., Kikuchi, M., and Ikehara, M. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 6903-6907) was analyzed by calorimetry and interpreted in terms of structural information obtained from x-ray crystallography. It was found that the enthalpic contribution for the Ca2+ binding reaction was small, driven primarily by entropy release (10 kcal/mol). This release of entropy was also observed in some organic chelators. Moreover, through the information of the tertiary structures of the apo- and holomutant lysozyme, it was confirmed that the entropy release (10 kcal/mol) upon the binding of Ca2+ arises primarily from the release of bound water molecules hydrating the free Ca2+. Previous studies of Ca2+ binding to proteins have involved significant changes in protein conformation. They can now be reevaluated to determine the contribution of conformational changes to Ca2+ binding. After removing the thermodynamic contribution of Ca2+ binding itself, it is found that upon the binding of Ca2+ the enthalpy change is negative but is almost compensated by the negative entropy change. The negative change in both enthalpy and entropy is characteristic of values seen in the thermodynamic change upon the folding of proteins.