Rotational dynamics of myosin heads in glycerinated cardiac muscle fibres were studied by using the spin-labelling technique in the conventional and saturation transfer electron paramagnetic resonance time domains. The maleimide spin labels which were attached to the fast-reacting thiols (SH1 or SH2, respectively) of the myosin moiety, exhibited orientational order with respect to the fibre axis in rigor state of the muscle fibres; the higher degree of order of the paramagnetic probe molecules was obtained when the labels were attached to the SH2 thiol sites. The degree of order of spin labels is reduced during muscle activity. In rigor the rotational correlation time of the labels bound to the SH2 sites was in the submillisecond time range (tau 2 approximately 30 microseconds), whereas tau 2 was equal to about 0.1 microseconds when the labels occupied the SH1 sites. The comparison of electron paramagnetic resonance spectra on cardiac and skeletal muscle indicates differences in the mobility of spin labels around the fast reacting thiols of the two myosins and also in the subpopulations of the labelled sites. On the basis of the rotational correlation times it can be suggested that a fraction of myosin heads might be free and/or the attached heads have some residual mobility or large degree of flexibility in rigor of the cardiac muscle at complete filament overlap.