Differential scanning calorimetry has been used to examine the thermal denaturation of rabbit hepatic galactoside binding protein. In the absence of Ca2+ or ligands, the inactive binding protein shows a single transition with a Tm of 46 +/- 0.5 degrees C and an enthalpy of denaturation of 0.891 cal g-1. In the presence of 20 mM CaCl2, the active binding protein has a single transition with a Tm of 61 degrees C and an enthalpy of denaturation of 2.67 cal g-1, indicating that Ca2+ markedly stabilizes the protein toward thermal denaturation. The Tm values of the binding protein--Ca2+ complexes with asialoorosomucoid or lactose are 64 and 63 degrees C, respectively. The enthalpy of denaturation in the presence of 20 mM lactose is 3.39 cal g-1, indicating that an additional stabilization (approximately 27%) toward denaturation is provided by binding of specific ligands. Furthermore, the differences in the shape of the denaturation profiles in the presence and absence of ligands suggest that ligand binding influences the denaturation process. Calcium binding, however, stabilizes the galactoside binding protein to thermal denaturation to a greater extent than does ligand binding. Thermal denaturation transitions attributable to the A or the B subunits of the binding protein are not observed, suggesting that the two subunits may be structurally similar.