Abstract The present communication reports on changes in the secondary and tertiary structures of native and apo-yeast alcohol dehydrogenase upon heating at 50 °C, as evident from circular dichroism (CD) studies. The presence of sugars provided significant protection with trehalose being the most effective. Exposure of hydrophobic clusters in the protein molecule upon heat denaturation was confirmed by fluorescence studies using 1-anilinonaphthalene-8-sulfonate (ANS) as a hydrophobic reporter probe. All sugars, and especially trehalose, reduced the affinity of both forms of the enzyme for this probe. The effectiveness of sugars in diminishing ANS fluorescence enhancement is in accordance with their ability to protect aggregation of the proteins, reported earlier [Miroliaei M, Nemat-Gorgani M. Sugars protect native and apo yeast alcohol dehydrogenase against irreversible thermoinactivation. Enzyme Microb Technol 2001;29:554–9]. It is concluded that prevention of the mechanisms of irreversible thermoinactivation may occur with retention of the secondary and tertiary structural properties of the proteins.