The stability of the substrate-binding region of human inducible Hsp70 was studied by a combination of spectroscopic and calorimetric methods. Thermal denaturation of the protein involves four accessible states: the native state, two largely populated intermediates, and the denatured state, with transition temperatures of 52.8, 56.2 and 71.2 degrees C, respectively, at pH 6.5. The intermediate spectroscopic properties resemble those of molten globules but they still retain substantial enthalpy and heat capacity of unfolding. Moreover, the similar heat capacities of the first intermediate and the native state suggests that the hydrophobic core of the intermediate would be highly native-like and that its formation would involve an increased disorder in localized portions of the structure rather than formation of a globally disordered state. The structure of the C-terminal of Hsp70 is destabilized as the pH separates from neutrality. The intermediates become populated under heat shock conditions at acidic and basic pHs. Denaturation by guanidine chloride also indicated that the protein undergoes a sequential unfolding process. The free energy change associated to the loss of secondary structure at 20 degrees C (pH 6.5) is 3.1 kcal.mol(-1) at high salt conditions. These values agree with the free energy changes estimated from differential scanning calorimetry for the transition between the second intermediate and the final denatured state.