Defferential scanning calorimetry was used to monitor the thermal transitions of the 70 kDa heat shock cognate protein (Hsc70). Hsc70 had endothermic trasitions with midpoints (Tm) at 59°C and 63°C in the absence and presence of ATP, respectively, and a similar increase in Tm was observed using intrinsic fluorescence of tryptophan. Combined with increased exposure at 60°C of non-polar residues of Hsc70 to which the hydrophobic, fluorescent probe ANS bound, these data indicate that the endotherms represent thermal denaturation and that bound nucleotide stabilizes Hsc70. An exothermic transition (Tm=66°C) was detected by calorimetry for Hsc70-apocytochrome c (apo c) complexes. An increase in intrinsic fluorescence with the same Tm and increased turbidity indicated aggregation of the denatured Hsc70-apo c. A novel finding was an exothermic transition of Hsc70 begining at about 30°c (Tm=41°C). No changes in either intrinsic fluorescence or ANS fluorescence attributable to protein transitions were detected in this temperature range. Examination of samples run on native polyacrylamide gels indicated that this exothermic transition was not due to Hsc70 aggregation or multimer formation. However, Hsc70 was protease-resistant at 20°C, sensitive at 40°C and resistant when returned to 20°C, indicating that this exotherm is associated with a reversible conformational change. As an assay for Hsc70 chaperoning function, complex formation was measured as a function of temperature using a variety of substrates including the model unfolded protein apo c a pigeon cytochrome c fragment, a representative hydrophobic-aromatic peptide FYQLALT, and a representative hydrophobic-basic motif NIVRKKK. For all of these substrates, the amount of complex formed increased with increasing termperature over the same range as the 41°C exotherm. It is proposed that a conformational change exposes polar and charged residues in Hsc70 Which subsequently become hydrated, resulting in an active chaperone. Hsc70 may be a thermal sensor that supply of chaperoning activity with demand for it over the physiological temperature range of mammalian cells. Thermal activation of Hsc70 may also have a role in acquired thermotolerance.