Lipase from Candida rugosa (CRL) has been purified and immobilized by using different immobilization protocols: interfacial adsorption on hydrophobic supports, ionic adsorption on PEI-coated supports, and covalent immobilization (on glutaraldehyde supports). This gave enzyme immobilized with different orientations and microenvironments. The catalytic properties (activity, specificity, and enantioselectivity) of the different derivatives have been found to be dramatically different. Very significant changes on activity with different substrates were found. For example, interfacially adsorbed derivative was the most active using simple substrates (ethyl butyrate) while PEI derivative was the most active hydrolysing ionic substrates (2-phenyl-2-butyroylacetic acid at pH 7) or methyl mandelate. The E value also depends strongly on the derivative and the conditions employed. Thus, the interfacially absorbed enzyme varied its enanatioselectivity (toward S isomer) from 1.6 to 85 in the hydrolysis of (R,S)-2-phenyl-2-butyroylacetic acid when the pH value varied from 7 to 5. However, the glutaraldehyde derivative presented a high enantioselectivity (E = 400) toward R isomer (the inverse E value compared to the previous derivative) at both pH conditions. Polyethyleneimine (PEI) derivative presented a slight enantiopreference toward the S isomer. Thus, using different derivatives, it has been possible to obtain both pure enantiomers from the ester or the product. Similar changes in the E values were obtained in the hydrolysis of methyl mandelate though here always there was a enantiopreference for the S isomer. Using this substrate, the best derivative was the PEI derivative at pH 5 (E = 300), while the glutaraldehyde one presented an E value of only 10.