An equilibrium dialysis study has revealed that porcine intestinal calcium-binding protein (CaBP) has two binding sites for Ca2+ whose dissociation constants (Kd) are the same, 0.56 microM. The intrinsic fluorescence spectrum of the CaBP shows a peak (at 303 nm) in tyrosine band. Ca2+ binding to the CaBP induces a monophasic increase in the intensity of intrinsic fluorescence without any shift to either excitation or emission maximum. The change in the fluorescence intensity induced by Ca2+ binding is complete at a bound Ca2+/CaBP molar ratio of about 2, and the apparent Kd value is 0.51 microM. The same bound Ca2+/CaBP molar ratio has been obtained from the maximal changes in UV absorption and CD spectrum of the CaBP upon addition of Ca2+. A CD study has shown an about 5% increase in alpha-helix content in the CaBP at the maximal binding of Ca2+. All these results indicate that the porcine intestinal CaBP has two high-affinity binding sites with equal affinity for Ca2+ and suggest that it undergoes a quantitative conformation change accompanying Ca2+ binding in the vicinity of single tyrosine and phenylalanine residues of the protein molecule.