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The soybean Kunitz trypsin inhibitor (SKTI) is a β-sheet protein with unusual stability to chemical and thermal denaturation. Different spectroscopic criteria were used to follow the thermal denaturation and renaturation of SKTI. Upon heating to 70°C, changes in UV difference spectra showed increased absorbance at 292 and 297nm, attributable to perturbation of aromatic residues. Cooling the protein resulted in restoration of the native spectrum unless reduced with dithiothreitol. Far- and near-UV CD spectra also indicate thermal unfolding involving the core tryptophan and tyrosine residues. Both CD and UV-absorbance data suggest a two-state transition with the midpoint at approximately 65°C. CD data along with the increased fluorescence intensity of the reporter fluorophore, 1-anilino-8-naphthalenesulfonate with SKTI, between 60 and 70°C, are consistent with a transition of the native inhibitor to an alternate …