Conventional and saturation transfer electron paramagnetic resonance spectroscopy (EPR and ST EPR) and differential scanning calorimetry (DSC) were used to study the motional dynamics and structural stability of cardiac myosins. Cardiac myosins isolated from bovine and human heart muscle were spin-labelled with a maleimide- or an iodoacetamide-based probe molecule at the reactive sulhydryl sites (Cys-697 and Cys-707). The probe molecules rotated with an effective rotational correlation time of 0.04 microsecond which was at least six times shorter than the rotational correlation time of the same label on skeletal myosin. In the presence of MgADP, flexibility changes in the multisubunit structure of myosins were detected, but this did not lead to changes of the overall rotational property of the myosin heads. Temperature dependence of the EPR spectra of maleimide spin-labelled myosin showed continuous decrease of the spectral parameters (amplitude ratio of the diagnostic peak heights in the ST EPR time domain and hyperfine splitting constant) at increasing temperature. In contrast, marked changes were obtained at about 17 degrees C in light chain-2 deficient myosin. DSC measurements supported the view that the removal of the light chain-2 produced additional loosening in the multisubunit structure of cardiac myosin. It is postulated that the light chain-2 is an integral part of myosin, and there is an intersite communication between light chain-2 and the 20 kDa subunit.