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 the 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.