The preparation and characterization of three trypsin-based monolithic immobilized enzyme reactors (IMERs) developed to perform rapid on-line protein digestion and peptide mass fingerprinting (PMF) are described. Trypsin (EC 3.4.21.4) was covalently immobilized on epoxy, carbonyldiimidazole (CDI) and ethylenediamine (EDA) Convective Interaction Media (CIM) monolithic disks. The amount of immobilized enzyme, determined by spectrophotometric measurements at 280nm, was comprised between 0.9 and 1.5mg per disk. Apparent kinetic parameters Km* and Vmax*, as well as apparent immobilized trypsin BAEE-units, were estimated in flow-through conditions using N-alpha-benzoyl-L-arginine ethyl ester (BAEE) as a low molecular mass substrate. The on-line digestion of five proteins (cytochrome c, myoglobin, alpha1-acid glycoprotein, ovalbumin and albumin) was evaluated by inserting the IMERs into a liquid chromatography system coupled to an electrospray ionization ion-trap mass spectrometer (LC-ESI-MS/MS) through a switching valve. Results were compared to the in-solution digestion in terms of obtained scores, number of matched queries and sequence coverages. The most efficient IMER was obtained by immobilizing trypsin on a CIM EDA disk previously derivatized with glutaraldehyde, as a spacer moiety. The proteins were recognized by the database with satisfactory sequence coverage using a digestion time of only 5min. The repeatability of the digestion (R.S.D. of 5.4% on consecutive injections of myoglobin 12microM) and the long-term stability of this IMER were satisfactory since no loss of activity was observed after 250 injections.