Gel filtration studies show that the thyroglobulin (Tg) molecule (dimer) binds from 18 to 50 Ca2+ ions. At pH 7.5 Tg binds 18 Ca2+ ions with a Kd of 1.3 x 10(-5) M, and 50 Ca2+ ions with a Kd of 5.5 x 10(-4) M. The binding of calcium to bovine thyroglobulin increases the absorption band of iodoamino acid residues at 315 nm. In the presence of Ca2+, the fluorescence intensity of 1-anilino-8-naphthalene sulfonate (ANS) is increased about 5-fold by Tg, with a shift in the fluorescence emission maximum from 505 to 490 nm. Thus, thyroglobulin possesses two classes of calcium binding sites with different affinities. The data reported indicate, also, that Ca2+ binding to Tg increases the hydrophobicity of the surface of the molecule.