An enantio- and regioselective hydrolytic kinetic resolution (HKR) of racemic p-chlorostyrene oxide (rac-pCSO) was achieved by epoxide hydrolase (EH) from recombinant Aspergillus niger in a selected neat organic solvent medium. The solid free EH was reused four times in repeated-batch reactors; however, the relative activity as well as the enantiomeric ratio (E-value) of this EH decreased from 100 to 20% and from 68 to 23, respectively, In order to overcome the diffusion hindrance, due to the accumulation of the hydrophilic diol in the enzyme micro-environment, and thereby to improve the operational stability of EH after recycling, strategies consisting of the immobilization of EH and the use of a binary organic solvent as the reaction medium were successfully applied. Although the highest protein immobilization yield (82%) and retention of EH activity (142%) in heptane were obtained upon the immobilization of EH on Accurel EP, the E-value and the operational stability of the resulted EH immobilizate after recycling were reduced as compared to the free EH. In contrast, the nonporous DEAE-cellulose improved the operational stability of EH by more than twofold. On the other hand, both the HKR efficiency and the operational stability of A. niger EH were found to be modest to excellent in various binary organic solvent mixtures of heptane and dioxane, depending on their ratio resulting in different Log A (C) 2009 Elsevier Ltd. All rights reserved.