The thermal unfolding of papain was studied at pH 2.6 by means of circular dichroism and difference spectroscopy. The transition curves obtained from ellipticity changes at 208 and 220 nm were biphasic, i.e., they showed two distinct successive steps, demonstrating the existence of an intermediate state of stable secondary conformation in the denaturation process. Difference-spectroscopy studies indicated that considerable exposure of aromatic side-chains is involved in both steps of the transition. Since papain has two domains in its molecular structure, our results suggest that they unfold in a successive way and rather independently. Furthermore, the structural characteristics of the intermediate state, obtained from its circular dichroism spectrum in the far-ultraviolet region, seem to point out that the second domain (residues 111-212) is the most stable part of the molecule.