Protease-catalyzed peptide synthesis in acetonitrile/water mixtures, containing 0-90% water, was investigated. alpha-Chymotrypsin, as well as thermolysin, were deposited on solid supports, prior to exposure to the reaction media. Peptide syntheses were performed using both a kinetically controlled process (chymotrypsin) and an equilibrium-controlled synthesis (thermolysin). The activity of chymotrypsin decreased at low water contents. However, at low water contents (1-10%) hydrolytic side reactions were suppressed and high yields of dipeptides were obtained. Optimal water content for the thermolysin-catalyzed reaction was 4-8%. The dipeptides produced were fully soluble in the reaction mixtures. High operational stability for alpha-chymotrypsin was obtained during 216 h of reaction.