The enzymatic hydrolytic resolution of (+/-)-glycidyl butyrate using a lipase from Candida antarctica (fraction B) (CAL-B) has been performed. This enzyme presented a significant enantioselectivity towards the R isomer, in contrast with the enantiopreference displayed in most lipases. The pure enzyme was immobilized using different protocols to obtain immobilized enzyme preparations with different orientations and microenvironments. The catalytic properties (activity, specificity, enantioselectivity) of the resulting immobilized preparations were found to be quite different from each other. Furthermore, the combination of these techniques with an appropriate reaction conditions permitted to obtain a quite pure (S)-glycidyl butyrate using as biocatalyst the CAL-B immobilized on a support covered with polyethylenimine at 25 degrees C, pH 7 and 10% of dioxane (ee > 90% at 64% of conversion). (c) 2005 Elsevier Inc. All rights reserved.