The thermal denaturation of bromelain, a cysteine proteinase from the papain family, was studied by means of circular dichroism (CD) and differential scanning calorimetry (DSC). It was found that this process is completely irreversible and apparently follows a simple two-state mechanism of the type N-->D. The activation energy, E, that characterizes this reaction was calculated by the use of different approaches: (i) the effect of heating rate on the temperature at which the transition is half completed; (ii) analysis of individual transition curves; (iii) kinetic studies at fixed temperatures; and (iv) single DSC tracings. The obtained values for E were rather similar to one another, varying from 164 to 226 kJ/mol. In comparison, the total calorimetric enthalpy change was 334 kJ/mol. When a more complex mechanism is considered (N<-->U-->D), which takes into account the presence of a reversibly unfolded state (U), our results suggest that the rate-limiting step is precisely the formation of U. Calculation of the corresponding activation enthalpy and entropy also seems to support this proposal.