High concentrations of proteins and enzymes have to be stored for extended periods of time. Under such conditions, at least three major factors contribute to aggregation and loss of protein function: hydrophobicity, propensity to form non-native beta-sheet structure and net charge of the polypeptide chain. Here we evaluate these thermal aggregation factors for horse liver ADH (alcohol dehydrogenase) and the effect of alpha-CyD (alpha-cyclodextrin) on the ADH aggregation, by using fluorescence, CD, UV-visible spectrophotometry, the DLS (dynamic light scattering) technique and the enzymatic activity assay. In addition, we propose the relative importance of the hydrophobic effect on the ADH aggregation. Although ADH readily forms aggregates at higher temperatures, alpha-CyD effectively diminishes this phenomenon. This reduction can be attributed to the prevention of the appearance of larger-size aggregated molecules and also to the higher homogeneity of the small nuclei under the alpha-CyD effect. The observed re-aggregation upon the addition of alpha-CyD/phenylalanine can be attributed to the competition binding of phenylalanine to the internal hydrophobic cavity of alpha-CyD. This signifies that aromatic amino acids are important regional components of the residual structure that may form nuclei for aggregation. The results of dynode voltage changes indicate that the thermal unfolding of ADH is accompanied by protein aggregation, which subsequently leads to irreversible thermal unfolding. Moreover, alpha-CyD causes thermal stabilization and delays the onset of secondary structural unfolding and aggregation by approx. 10 degrees C and the midpoint ('melting') temperatures (T(m)) by more than 5 degrees C. Furthermore, alpha-CyD diminishes the deactivation of the enzyme, decreasing the deactivation constant by more than 50%, and clearly reveals the stabilization of the enzyme not only structurally but also kinetically at higher temperatures.