Differential scanning calorimetry (DSC) was applied to investigate the thermal unfolding of rabbit skeletal muscle G-actin in its complexes with actin-binding proteins, cofilin, twinfilin, and profilin. The results show that the effects of these proteins on the thermal stability of G-actin depend on the nucleotide, ATP or ADP, bound in the nucleotide-binding cleft between actin subdomains 2 and 4. Interestingly, cofilin binding stabilizes both ATP-G-actin and ADP-G-actin, whereas twinfilin increases the thermal stability of the ADP-G-actin but not that of the ATP-G-actin. By contrast, profilin strongly decreases the thermal stability of the ATP-G-actin but has no appreciable effect on the ADP-G-actin. Comparison of these DSC results with literature data reveals a relationship between the effects of actin-binding proteins on the thermal unfolding of G-actin, stabilization or destabilization, and their effects on the rate of nucleotide exchange in the nucleotide-binding cleft, decrease or increase. These results suggest that the thermal stability of G-actin depends, at least partially, on the conformation of the nucleotide-binding cleft: the actin molecule is more stable when the cleft is closed, while an opening of the cleft leads to significant destabilization of G-actin. Thus, DSC studies of the thermal unfolding of G-actin can provide new valuable information about the conformational changes induced by actin-binding proteins in the actin molecule.