The unfolding of rabbit muscle-type creatine kinase (MM-CK) induced by guanidine hydrochloride (GuHCl) has been studied by isothermal microcalorimetry. It has been found that the decrease in the activity of NIM-CK in dilute GuHCl solution is due to a slight perturbation of the active site conformation by dilute GuHCl, but not by a reversible inhibition by GuHCl binding at the active site or dissociation of the dimeric protein. The inactivation of MM-CK precedes the overall conformation change of this enzyme during denaturation by GuHCl, providing a thermodynamic evidence for the proposition that the active site of an enzyme is situated in a limited region more flexible than the enzyme molecule as a whole. The intrinsic enthalpy, Gibbs free energy, and entropy changes for formation of an intermediate state of NIM-CK in the presence of moderate GuHCl concentrations at 25.00 degreesC have been determined to be 260, 12.2 kJ mol(-1), and 830 J mol(-1) K-1, respectively. Further unfolding of MM-CK is observed when GuHCl concentration is higher than 3.00 mol dm(-3), and the protein is almost fully unfolded at 5.00 mol dm(-3) GuHCl reached. The intrinsic enthalpy, Gibbs free energy, and entropy changes for formation of the unfolded state of MM-CK at 25.00 degreesC have been measured as 8600, 23.0 kJ mol(-1), and 29 kJ mol(-1) K-1, respectively. The experimental results indicate that the unfolding of MM-CK by GuHCl exhibits remarkable enthalpy-entropy compensation and the water reorganization is involved in the unfolding reaction.