The structure and conformations of pig intestinal Ca-binding protein (CaBP) have been studied by terbium luminescence enhancement and circular dichroism. The two cation-binding sites bind Tb3+ sequentially; the affinity of the first site is greater than 10(7) M-1 and the affinity of the second site is approximately 10(5) M-1. Filling of the first site enhances the fluorescence of the single tyrosine residue, whereas Tb3+ in the second site quenches the fluorescence. Excitation spectra of the Tb3+-bound forms of CaBP show that considerable energy transfer takes place from phenylalanine residues to the bound Tb3+, although some transfer from tyrosine is also detected. The sequence in which the sites are filled was deduced from these results and the published three-dimensional structure of the cow intestinal CaBP. Tb3+ bound approximately 20 A (1 A = 0.1 nm) from the tyrosine induced a large increase in the optical activity of this residue. We argue that a potentially important conformational change is induced in CaBP by cation binding.