High-sensitivity differential scanning calorimetry has been applied to the study of the thermal denaturation of thermolysin from Bacillus thermoproteolyticus rokko and its 255-316 and 205-316 fragments. Stability investigations into thermolysin have been extended from a previous calorimetric study at pH 7.5 [2] to different experimental conditions, which included 0.3-3.7 mg/ml of protein concentration, pH values within the range 3.0-9.0, and inhibitors such as phosphoramidon and 1,10-phenanthroline. The thermal transitions were always irreversible, kinetically controlled and followed the two-state kinetic model. Autolysis of native thermolysin and/or the unfolded enzyme together with aggregation of the unfolded state in the presence of inhibitors seem to be;he reasons for the irreversible denaturation. On the other hand, calorimetric studies into the concentration effects on the 255-316 and 205-316 thermolysin fragments show the presence of dimers in solution undergoing equilibrium unfolding processes. The thermodynamic parameters of unfolding for both fragments are consistent with a higher compact globular structure for the shorter dimeric fragment than for the larger one. Given their spontaneous folding capability, these fragments could well be considered as folding domains in thermolysin.