Silica and polyacrylamide microspheres were modified with chemisorbed chymotrypsin and used to enzymatically hydrolyze a peptide thin film which was covalently bound to a flat silica surface. Chymotrypsin was covalently cross-linked to 500 nm silica spherical beads and 30-50 mu m polyacrylamide spherical beads and shown to be enzymatically active against thin films of a fluorescent peptide, succinyl-ala-ala-phe-7-amido-4-methylcoumarin (SAAP-AMC), and an unlabeled peptide, t-BOC-phe. SAAP-AMC and t-BOC-phe were covalently coupled to an aminosilane film on silica and aluminum substrates through an amide linkage. Control experiments showed that free chymotrypsin in solution was able to hydrolyze the amide bond between the phenylalanine and the AMC groups of the chemisorbed peptide, resulting in the release of the AMC group into solution. When they were placed into contact with the SAAP-AMC surface, the chymotrypsin-modified beads also hydrolyzed the phe-AMC bond and released AMC into solution, demonstrating that covalently immobilized enzymes can be used to hydrolyze immobilized organic thin films. The hydrolytic activity of the chymotrypsin beads was also confirmed for a second peptide film, t-BOC-phe, by external reflectance IR spectroscopy.