A characteristic property of the tryptophan synthase α₂β₂ complex is the mutual activation of the α and β subunit upon complex formation. It has been speculated that this mutual activation results from the conformational change due to the α/β subunit interaction. In order to elucidate this mechanism, the association for the various combinations of the α and β subunits from mesophiles, Escherichia coli and Salmonella typhimurium, and for the two subunits from a hyperthermophile, Pyrococus furiosus, was examined using isothermal titration calorimetry. In mesophile proteins, the analyses of the thermodynamic parameters of association indicate that both the α and β subunits fold coupled with the association and the folding might occur not only at the subunit contact surface but also at the other parts in the molecules. This conformational rearrangement might be the origin of mutual activation. The thermodynamic measurements also revealed that the substitution of only one residue in the subunit interface of the E. coli a subunit changed the thermodynamic properties of association with the β subunit similar to those of the S. typhimurium α subunit. On the other hand, the association between α and β₂ subunit from P. furiosus was characterized by substantially low values of association enthalpy and association heat capacity changes. This suggests that the folding coupled with the association decreases at low temperatures around 40 ℃ examined. This relates with remarkably low activities around 40 ℃, as compared with those of mesophile proteins. These differences in thermodynamic properties were discussed on the basis of X-ray structures of both proteins.