Thermophilic malate dehydrogenase [L-malate:NAD+ oxidoreductase, EC 1.1.1.37] was denatured at pH 2.0 with complete loss of enzyme activity but without dissociation to monomers, suggesting the presence of strong intersubunit contact. On the other hand, the enzyme was completely denatured and dissociated to monomers in the presence of 5 M GdnHCl. Inactivation and denaturation of the enzyme by acid and GdnHCl were reversible. Upon dilution of the denaturants, the inactivated enzyme regained enzyme activity and the native structure with high yield (80-90%). Kinetic analyses of reactivation of the enzyme denatured by GdnHCl and by acid revealed that the reaction obeyed first-order kinetics. The rate constant and Arrhenius activation energy of the reactivation of the acid-inactivated enzyme were almost the same as those of the enzyme inactivated by GdnHCl. These results suggest that the rate-limiting steps in the reactivation processes of the enzyme denatured by GdnHCl and by acid are the same and that a conformational change of the inactive dimer to active dimer is the rate-limiting step in the reactivation reaction.