Active uniform films of horseradish peroxidase (HRP) have been prepared by covalent binding on Si/ SiO2 or glass supports previously activated by silanization and succinylation. Labeling by fluorescent or by Electron Spin Resonance (ESR) probes was used to quantify the surface density of active groups and of horseradish peroxidase. Atomic Force Microscopy (AFM) imaging was used to characterize the surface morphology. We observed that a non-uniform protein adsorption due to physical interactions was present when the supports were not activated for covalent binding and was, in large part, removed by washing. The enzyme deposited by covalent binding formed homogeneous layers with a height in the range 60-90 Angstrom. By using a fluorescent label, we calculated a protein density of 3.6 x 10(12) molecules cm(-2) on Si/SiO2, corresponding to an estimated area per molecule of 2800 Angstrom(2) which is in agreement with the value expected on the basis of the crystallographic data considering the formation of a monomolecular layer. The protein density of the layer immobilized on glass was similar (1.9 x 10(12) molecules cm(-2)). The enzyme immobilized on both supports showed a k(cat)/K-M being of the order of 3-5x10(5) M-1 s(-1) that is 1/20th of free HRP. The half-life time of the activity of the enzyme immobilized by covalent binding was longer than 40 days at 6 degrees C. (C) 2000 John Wiley & Sons, Inc.