Effects of mutations of a protein, Streptomyces subtilisin inhibitor, on cold denaturation have been examined by means of differential scanning calorimetry. Cold denaturation was observed in a mutation-independent manner, while heat denaturation was clearly dependent on the mutations, suggesting that hydration of polar peptide bonds are responsible for the transition of a protein structure at low temperature. The hydration effect was also examined by extrapolating thermodynamic data previously obtained for amino acid replacements at Met73, which is completely exposed to solvent. Enthalpic changes induced by the mutations were shown to be nearly compensated by the entropic changes at 25 degrees C, whereas the deviation became larger at 100 degrees C, suggesting that the hydration is a primary cause for the compensation.