A new approach is proposed to immobilize lipase into microporous polymeric membrane via embedding cross-linked enzyme aggregates (CLEA) within membrane pores. Preparation of biocatalytic membranes was implemented with hydrophilic cellulose and hydrophobic PTFE membranes. Formation of lipase CLEAs is initiated by solvent exchange between aqueous solution of the enzyme filling membrane pores and organic media containing cross-linking agent. The influence of organic media composition and matrix hydrophilicity/hydrophobicity on membrane biocatalytic properties has been studied. Highly effective biocatalytic membranes with embedded lipase CLEA have been obtained on the base of both hydrophilic and hydrophobic membranes by varying the content of glutaric dialdehyde used as a cross-linking agent. Bio-imprinting has been tested as a mean to improve efficiency of the biocatalytic membranes. Biocatalytic esterification of oleic acid with n-butanol has been studied in cross-flow membrane bioreactor with membrane-supported lipase CLEA using hexane and iso-octane as reaction solvents of different hydrophobicity. (C) 2004 Elsevier B.V. All rights reserved.