A dipeptide synthesis was drastically influenced by the reaction temperature, in the range from -30 degrees to 25 degrees C. This article shows the kinetic reasons of this effect. alpha-Chymotrypsin was immobilized on celite and used in four different water-miscible solvents containing small amounts of water. The reaction studied was the aminolysis of N-acetyl-L-phenylalanine ethyl ester (Ac-PheOEt) with L-alaninamide (Ala-NH2). In the competition between the nucleophile (Ala-NH2) and water for the acylenzyme complex, the nucleophile was favored by low reaction temperatures. This effect (quantified as p-values) was observed in all four solvents, and it was greatest in acetonitrile and tetrahydrofuran. The esterase and amidase activities of the enzyme were studied using Ac-PheOEt and N-acetyl-L-phenylalanyl-L-alaninamide (Ac-PheAla-NH2) as substrates. The Michaelis-Menten parameters, K-m,K-app and V-max, were determined for ester hydrolysis and dipeptide hydrolysis. Both K-m,K-app and V-max tended to increase with increasing temperature. Secondary hydrolysis was reduced at subzero temperatures because ester hydrolysis was favored in relation to dipeptide hydrolysis. Dipeptide synthesis was thus favored by low temperatures in two ways : first, in the competition between the nucleophile and water for the acyl enzyme; and, second, in the competition between the ester substrate and the peptide substrate for the free enzyme. As a result, in acetonitrile containing 10% water, the maximal yield was 99% at -20 degrees C compared with 84% at 25 degrees C.