The thermodenaturation of phosphoenolpyruvate carboxylase (PEPC) from an extremely thermophilic bacterium Rhodothermus obamensis, capable of growth at temperature np to 85 degrees C, was probed at different denaturation temperatures by UV-visible absorption, fluorescence emission and 1-anilinonaphthalene-8-sulfonate (ANS) binding and renaturation was assessed from different states of denaturation. Under severe denaturation conditions at 100 degrees C, the enzyme was rapidly inactivated and its global structure immediately reached the irreversibly aggregated state by passing through the dissociated and the putative scrambled states as observed by UV-visible absorption spectroscopy. However, under milder conditions of denaturation at 93 degrees C, the enzyme was gradually inactivated, and its global structure shifted sequentially from the dissociated state to the scrambled state. At 80 degrees C, about 50% of the activity was left and no apparent change in the global structure occurred even after 30 h. In addition, ANS binding to the enzyme was greatly increased in accordance with the change in global structure. This implies that the hydrophobic regions of the enzyme tend to be exposed to solvent due to thermal dissociation and unfolding. The extrinsic thermostabilization factors that enhance the thermostability of the enzyme successfully suppress the thermodenaturation of the enzyme, especially the dissociation of its tetrameric form. Of these factors, the substrate for the enzyme, phosphoenolpyruvate (PEP), causes the reassociation of the dissociated inactive form of the enzyme to the active form. These results suggest that the global thermodenaturation of the enzyme results from the temperature-dependent shift of three different states and that the extrinsic thermostabilization factors act to a large extent to maintain quaternary structure.