The thermal unfolding of myosin in skeletal muscle myofibrils was studied by differential scanning calorimetry (DSC). In the absence of nucleotide two major transitions with Tm of 52 degrees C and 58 degrees C, and a minor transition with Tm of 19 degrees C were detected. The unfolding can be characterized with a total enthalpy of -90 +/- 6.1 mJ/g protein. The major transition with Tm of 58 degrees C was independent of the presence of nucleotide and orthovanadate (Vi), and it can be assigned to the unfolding of the alpha-helical rod part of myosin and partly to actin. In the presence of MgADP, the minor transition shifted to higher temperature, indicating changes between the heavy chain of subfragment-1 and the LC-2 light chain. The transition with Tm of 52 degrees C exhibited a significant broadening and a small shift to lower temperature. It indicates an internal domain or segmental rearrangement of the myosin motor. Upon addition of MgADP and Vi, a shift to higher temperature was observed for the lower major transition, evidencing that with trapped ADP and Vi the intermolecular interactions stabilized the myosin head region.