The thermal stability of actin isolated from skeletal muscle was studied in monomer and polymerized forms using DSC and EPR spectroscopy, Actin was labelled with the paramagnetic derivative of maleimide at the reactive thiol site Cys-374 in the C-terminal subdomain-1. The unfolding of actin induced by heating in the range 10-70 degrees C resulted in a single transition : the transition temperature was 51 +/- 2 degrees C for G-actin, whereas 63 +/- 2 degrees C was obtained for the transition temperature of filamentous actin. Assuming a simple two-state transition for F-actin, the van’t Hoff enthalpy was calculated to be 343.6 +/- 25.1 kJ mol(-1). The spectral changes were reversible in the temperature interval 10-65 degrees C for F-actin, and actin did not lose its ability to polymerize. The analysis of the DSC profiles for actin in monomer and polymerized forms enabled the assignment of three endothermic components in the F-form (T-ml = 59.7 +/- 1.4 degrees C, H-1 = 60.3 +/- 1.7 kJ mol(-1); T-m2 = 60.6 +/- 1.6 degrees C, H-2 = 348.2 +/- 9.6 kJ mol(-1); T-m3 = 61.4 +/- 1.4 degrees C, H-3 = 104.3 +/- 4.6 kJ mol(-1)), and two components in the G-form (52.5 +/- 1.4 and 56 +/- 1.8 degrees C with enthalpies 184.4 +/- 10.9 and 110.2 +/- 7.5 kJ mol(-1). Comparison of the spectroscopic and calorimetric behavior of actin showed remarkable differences in transition temperature and enthaply in both forms of actin, suggesting intramolecular and intermolecular interactions between subdomains and monomers that stabilize actin, especially in filament form.