S-layer ultrafiltration membranes (SUMs) with an active filtration layer composed of coherent two-dimensional, isoporous protein crystals (S-layers) have been used as matrix for immobilizing monolayers of enzymes. Since S-layers are formed by periodic repetition of identical protein subunits, functional groups are present on the crystalline array in an identical position and orientation. As a consequence monolayers of enzymes can bind in a geometrically well defined way. For the covalent immobilization of enzymes carboxyl groups from the S-layer protein were activated with carbodiimide and allowed to react with amino groups of the enzyme. SUMs were employed as a new type of immobilization matrix for the developement of an amperometric glucose sensor using glucose oxidase (GOD) as the biologically active component. Glucose oxidase covalently bound to the surface of the S-layer protein retained approximately 40% of its activity. The enzyme loaded SUMs were covered with a layer of gold or platinum to function as working electrodes. These sensors yielded high signals (150nA/mm2/mmol glucose), fast response times (10–30s) and a linearity range up to 12 mM glucose. The stability under working conditions was more than 48 hours. There was no loss in activity after a storage period of 6 month.