Immobilisation of urease onto plain and six-carbon spacer-arm (i.e. 1,6-diaminohexane) attached poly(hydroxyethyl methacrylate-co-glycidyl methacrylate) [poly(HEMA-GMA)] films was studied. The epoxy groups containing poly(HEMA-GMA-1-3) films were prepared by UV-initiated photopolymerisation of 2-hydroxyethyl methacrylate (HEMA) and glycidyl methacrylate (GMA). The spacer arm (i.e. 1,6-diaminohexane) was attached through the epoxy groups of the films. Urease was then covalently immobilised either on the plain or on the spacer-arm-attached poly(HEMA-GMA-2) films using glutaric dialdehyde as a coupling agent. Incorporation of the spacer-arm resulted in an increase in the apparent activity of the immobilised urease with respect to the enzyme immobilised on the plain films. The urease immobilisation capacity of the films was increased as the GMA ratio increased in the film structure. The activity yield of the immobilised urease on the spacer-arm-attached poly(HEMA-GMA-2) films was 53%, and 36% for the enzyme immobilised on the plain poly(HEMA-GMA-2) films. Both immobilised urease preparations have resistance to temperature inactivation when compared to that of the free form. The temperature profiles were broader for both immobilised preparations than that of the free enzyme. Kinetic parameters were determined for immobilised urease preparations as well as for the free enzyme. The values of the Michaelis constants Km for all the immobilised urease preparations were significantly larger, indicating decreased affinity by the enzyme for its substrate, whereas Vmax values were smaller for both immobilised urease preparations. In an 80-h continuous operation with spacer-arm-attached poly(HEMA-GMA-2) films at 35°C, only 7% of immobilised urease activity was lost. The operational inactivation rate constant (kopi) of the immobilised urease was 1.51 × 10-5 min-1. © 2003 Elsevier B.V. All rights reserved.