Different methods were evaluated to immobilise Pig Liver Esterase (PLE) in hollow fibre membranes. Four covalent bonding techniques (using epoxy, imidazol, amino and carboxylic acid terminal groups) were tested to link the enzyme to microporous nylon membranes. Physical immobilisation was also studied, by entrapment of the enzyme inside the microporous structure of a polysulfone asymmetric ultrafiltration membrane. The entrapment method lead to a higher retention of enzymatic activity for a longer period of time. This technique was selected to be used in a biphasic membrane bioreactor where the microporous hydrophilic membrane, containing the enzyme, is used to separate an aqueous from an organic phase, in which the substrate is dissolved. Different enzyme loading procedures were studied in the biphasic reactor and the resulting axial and radial enzyme distribution in the hollow fibre module were related to the global enzymatic activity.