We investigated preferential interactions of alpha-chymotrypsin with water-ethanol mixtures at 25 degrees C. Our approach is based on the analysis of residual enzyme activity and water/alcohol sorption. There are three concentration regimes. alpha-Chymotrypsin is preferentially hydrated at high water content. The residual enzyme activity is close to 100%. alpha-Chymotrypsin has a higher affinity for alcohol than for water at intermediate water content. Residual enzyme activity is close to zero in this concentration range. At low water content, ethanol is preferentially excluded from the protein surface. This results in preferential hydration of alpha-chymotrypsin and significant residual catalytic activity (similar to 50%) in water-poor ethanol. (C) 2017 Elsevier B.V. All rights reserved.