Thermal stability of actin, isolated from skeletal muscle, was studied in monomeric and polymerized form using DSC and EPR spectroscopy. The analysis of the DSC profiles in both forms enabled the assignment of the components : two endothermic components in the G-form (47.3+/-1.4 degrees C and 53.4+1.8 degrees C with enthalpies of 184.4+/-10.9 and 120.0+/-7.5 kJ/mol), and three endothermic components in the F-form (T-m1=59.7+/-1.4 degrees C, H-1=171.3+/-1.7 kJ/mol; T-m2=60.6+/-1.6 degrees C, H-2 - 231.2 +/- 9.6 kJ/mol; T-m3=61.3+/-1.4 degrees C, H-3=148.3+/-4.6 kJ/mol). Actin was labeled with the paramagnetic derivative of maleimide at the reactive thiol site Cys-374 in the C-terminal subdomain-1. The rotational correlation time for MSL-F-actin is 100 ys, as obtained from the peak ratio L "/L=0.745+/-0.05 (n=6), which indicates that there is significant internal flexibility in F-actin. The unfolding of actin induced by heating resulted in a single transition in both forms of actin. The comparison of G- and F-actins showed remarkable differences in transition temperature and enthalpy, suggesting intramolecular interactions between subdomains and monomers.