Differential scanning calorimetry (DSC) was used in conventional and temperature-modulated mode to study the energetics of myosin in skeletal muscle fibres in different states of the actomyosin ATPase cycle. Psoas muscle fibres from rabbit were used in the experiments with and without the presence of nucleotides (ATP, ADP, AMP-PNP) and ATP or ADP + orthovanadate. In the complex DSC pattern, the higher transition referred to the head region of myosin. The enthalpy of the thermal unfolding depended on the nucleotides, the conversion from a strongly attached state of myosin to actin to a weakly binding state was accompanied with an increase of the transition temperature which was due to the change of the affinity of nucleotide binding to myosin. This was more pronounced in TMDSC mode, indicating that the strong-binding state and rigor state differ energetically from each other. The different transition temperatures indicated alterations in the internal microstructure of myosin head region. The monoton decreasing TMDSC heat capacities show that the C-p of biological samples should not be temperature independent.