Temperature-gradient gel electrophoresis (TGGE) has been used to study the thermal unfolding of ferricytochrome c in low and high concentrations of acetic acid. It has been observed that the mobility of cytochrome c is a linear function of temperature when the system is characterized by a homogeneous population of conformation-state, single molecular species. Within the transition temperature range, the mobility clearly displays the characteristic sigmoidal shape describing the transitions of protein unfolding. The data obtained by TGGE were used to estimate the apparent thermodynamic parameters (enthalpy change Delta H-vh and transition temperature T-m), associated with the transition of unfolding. The accuracy of the apparent thermodynamic parameters obtained by this method agrees within error limits with the values obtained by direct calorimetric measurements using differential scanning calorimetry (DSC).