Because of its low cost, chemical and mechanical properties and ready availability in a number of different forms (e.g. powders, beads, nets, tubes, film, sheets, etc.) Nylon is an attractive matrix for enzyme immobilization. We report here a thorough evaluation of a protocol for enzyme immobilization on nylon film with relatively inexpensive and non-toxic reagents, involving acid hydrolysis, glutaraldehyde coupling and spacer molecules and employing beta -glucosidase and trypsin as model enzymes. We also describe steps for virtually eliminating enzyme leakage and non-specific binding. Individual steps in the procedure are simple and conditions flexible so, whilst evaluated in terms of binding proteins to nylon film, they should be applicable to other forms of nylon and suitable for binding most enzymes and proteins, including antibodies, providing a method having potential in both affinity chromatography/adsorption and in bioreactor applications. (C) 2001 Elsevier Science Inc. All rights reserved.