The thermal denaturation of azurin in H 2 O, D 2 O and in ethanol-H 2 O mixtures has been investigated by electron spin resonance (ESR), optical absorption spectroscopy and differential scanning calorimetry (DSC). The OD 6 2 5 /T variation observed at a scan rate of 0.7°C/min in H 2 O shows a cooperative OD transition between 78 and 82°C. In this step the intense charge-transfer band of azurin at 625 nm disappears. The ESR spectra recorded at -153°C of the protein in the native state and after heating at 80 and 82°C indicate that both the symmetry and the copper ligands change with the thermal transition. The DSC measurements show that the thermal denaturation of azurin, which occurs at 84.4°C, is irreversible and kinetically controlled. This complex transition has been described as a multistep denaturation path and was analysed using a Lumry-Eyring type mechanism. The experimental C p , e x c profile has been simulated and the calorimetric enthalpies related to the reversible and irreversible step, ΔH u and ΔH a g , respectively, are obtained. The kinetically controlled steps have been investigated by means of optical and DSC measurements at different scan rates and the apparent activation energy, E a , has been calculated. The denaturation of azurin in D 2 O and ethanol-H 2 O mixtures follows the same denaturation path as in H 2 O, although a shift of the OD 6 2 5 /T and DSC profiles is evidenced. The temperature of the thermal transition and theE a values decrease in ethanol-H 2 O mixtures, but increase in D 2 O.