We study the application of hierarchical ZSM-5 zeolites, combining micropores and intracrystalline mesopores, as carriers for lipase enzymes compared with purely microporous ZSM-5 and mesoporous MCM-41. Strategies to improve enzyme immobilization by modification of the support porosity and surface properties (e.g. by reaction with organosilane, or by treatment with the enzyme cross-linking agent glutaraldehyde) are also reported. Spectroscopic screening of catalyst activity and recyclability for the aqueous phase hydrolysis of p-nitrophenyl esters, permits evaluation of the influence of support properties and immobilization conditions on the performance of the resulting biocatalysts. An excellent correlation is observed between the mesopore surface area, the enzyme uptake, and the corresponding biocatalyst activity, demonstrating the functional character of mesopores in hierarchical zeolites. Modification of the mesopore walls prior to enzyme immobilization is essential to attain an active and recyclable biocatalyst. Enzymes immobilized on purely inorganic supports exhibit rapid loss of activity attributed to enzyme leaching. Despite the high mesopore surface area of surface-functionalized MCM-41, the mono-dimensionality of the mesopores results in restricted accessibility and a reduced enzyme uptake. In comparison, the interconnected mesopores of the hierarchical zeolites remain accessible after surface functionalization showing good adsorption properties. Lipase immobilized on thiol-functionalized mesoporous ZSM-5 was found to be the most efficient biocatalyst. (C) 2010 Elsevier B.V. All rights reserved.