Troponin-C (TnC) from rabbit skeletal muscle contains two high affinity Ca2+ binding sites (sites 1 and 2) that bind Mg2+ competitively (Ca2+-Mg2+ sites) and two Ca2+ binding sites (sites 3 and 4) of lower affinity that do not bind Mg2+ (Ca2+-specific sites). The free energy (deltaG0i), enthalpy (delta H0i) and entropy (deltaS0i) of binding Ca2+ to each of the four sites (i = 1 to 4) on TnC have been evaluated from microcalorimetry and equilibrium dialysis. The enthalpy of Ca2+ binding to each site was identical (-7.7 kcal mol-1); the entropy of Ca2+ binding to sites 1 and 2 was deltaS01,2 approximately equal to 14.7 e.u. whereas delta S03.4 approximately equal to 8.0 e.u. The positive entropy associated with Ca2+ binding to sites 1 and 2 is probably due to displacement of water produced by the alpha-helix formation, known to accompany the binding of Ca2+ to the Ca2+-Mg2+ sites. Thus, Ca2+ binding to the Ca2+-Mg2+ sites is driven by both enthalpy and entropy and the lower Ca2+ affinity for sites 3 and 4 is reflected in the lower entropy of Ca2+-binding. The entropy associated with Ca2+ binding to sites 3 and 4 suggests that some change in protein conformation is occurring upon binding of Ca2+ to these sites.