Microcalorimetic titrations were carried out to measure the thermodynamic functions of bullfrog skeletal muscle troponin C (TnC) in the interaction with Ca2+ and Mg2+, at 25 degrees C and at pH 7.0. Enthalpy titration curves with Ca2+ were composed of three stages both in the presence and in the absence of Mg2+. The first (0-2 mol Ca2+/mol TnC) and the third (greater than 3 mol Ca2+/mol TnC) stages were exothermic and the second stage (2-3 mol Ca2+/mol TnC) was endothermic. Mg2+ affected the first stage to decrease the amount of heat produced but not the second and third stages. The enthalpy titration with Mg2+, in the absence of Ca2+, was slightly exothermic initially and then became endothermic beyond 2-3 mol Mg2+/mol TnC. Absorption of heat was observed throughout the additions of Mg2+ in the presence of 1 mM Ca2+. The results indicate that bullfrog TnC has two high-affinity Ca2+-Mg2+ sites, two low-affinity Ca2(+)-specific sites, and two or around two Mg2(+)-specific sites. Based on the enthalpy and entropy changes, the Ca2+ binding reactions of TnC were classified into three types, indicating thermodynamic variety in the binding sites of the molecule.