Organophosphorus hydrolase (OPH), immobilized within polyelectrolyte multilayers deposited on glass beads (30-50 mum), showed sustained catalytic activity under ambient conditions for several months. OPH multilayers consist of two priming layers, branched poly(ethylene imine) (BPEI) and polystyrenesulfonate (PSS), followed by five alternating layers of BPEI and OPH. OPH multilayers coated on glass beads demonstrate catalytic activity similar to that of free enzyme, but over a much larger time scale (i.e., months vs hours). Stability of OPH multilayers coated on glass beads were also evaluated after depositing poly(acrylic acid) (PAA) as an outer priming layer for monomer assembly via electrostatic interactions. Three polymerizable monomers, 1,2-dihydroxypropyl methacrylate (DHPM), 1,2-dihydroxypropyl 4-vinylbenzyl ether (DHPVB), and N-[3-(trimethoxysilyl)propyl]ethylenediamine (TMSED), were anchored to the outer PAA layer and subsequently polymerized by UV irradiation or base-catalyzed hydrolysis. Polymerized DHPVB and TMSED rendered robust multilayer systems without affecting OPH activity. In aqueous sodium chloride (2 M for 2 h), OPH multilayers stabilized with an outer coating of TMSED remained 27% active as opposed to 0% activity for uncapped systems.