For the first time, the hydrolytic resolution of (R,S)-ethyl 2-chloromandelate in biphasic media by using hydrolase of Klebsiella oxytoca as a promising biocatalyst is reported for producing the desired (R)-ethyl 2-chloromandelate as an important intermediate for the synthesis of Clopidogrel. Effects of various operating parameters on the enzyme activity and enantioselectivity were systematically studied, from which the optimal condition of using isooctane as the organic phase, enzyme concentration of 2 mg/ml, temperature of 55 degrees C, and pH 6 of the aqueous phase was selected. The kinetic constants k(2R) = 4.6 x 10(-3) mmol/h g, k(2S) = 18.2 mmol/h g, k(mR) = 0.72 mM, k(mS) = 0.90 mM, and K-I = 65.5 mM were further estimated from the kinetic analysis at the optimal condition. By considering the acid product inhibition and non-enzymatic hydrolysis for the enzymatic resolution, good agreements of the time-course conversions from experiments and the theoretical prediction were also obtained. (c) 2006 Elsevier Inc. All rights reserved.