Calcium-binding proteins play various and significant roles in biological systems. Conformational changes in their structures are closely related to their physiological functions. To understand the role of calcium-binding protein 3 (CBP3) in Dictyostelium discoideum, its recombinant proteins were analyzed using circular dichroism (CD) and fluorescence spectroscopy. Gel mobility shift analysis showed that Ca 2+ induced a mobility shift of the recombinant CBP3. Far ultra-violet CD spectra and intrinsic fluorescence spectra on CBP3 and its N-and C-terminal domains exhibited that they underwent a conformational rearrangement depending upon Ca 2+ binding. Measurement of Ca 2+ dissociation constants demonstrated that CBP3 had high affinity toward Ca 2+ in the sub-micromolar range and N-terminal domain had higher affinity than C-terminal domain. The changes of fluorescence spectra by an addition of 8-anilino-1-naphthalene sulfonic acid indicated that the hydrophobic patches of CBP3 and its C-terminal domain are likely to be more exposed in the presence of Ca 2+ . Since the exposure of hydrophobic patches is thermodynamically unfavorable, Ca 2+ -bound CBP3 may interact with other proteins in vivo. All these data suggest that Ca 2+ induces CBP3 to be more favorable conformation to interact with target proteins.