In this paper, we reported the construction of a hybrid biosensor for direct, highly selective, sensitive, and rapid quantitative determination of organophosphate pesticides with p-nitrophenyl substituent using purified organ ophosphorus hydrolase (OPH) for the initial hydrolysis and Arthrobacter sp. JS443 for subsequent p-nitrophenol oxidation. The biocatalytic layer was prepared by coimmobilizing Arthrobacter sp. JS443 and OPH on a carbon paste electrode. OPH catalyzed the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon and methyl parathion to release p-nitrophenol that was oxidized by the enzymatic machinery of Arthrobacter sp. JS443 to carbon dioxide through electroactive intermediates 4-nitrocatechol and 1,2,4-benzenetriol. The oxidization current of the intermediates was measured and correlated to the concentration of organophosphates. The best sensitivity and response time were obtained using a sensor constructed with 0.06 mg dry weight of cell and 965 IU of OPH operating at 400 mV applied potential (vs. Ag/AgCl reference) in 50 mM citrate-phosphate pH 7.5 buffer at room temperature. Using these conditions, the biosensor measured as low as 2.8 ppb (10 nM) of paraoxon and 5.3 ppb (20 nM) of methyl parathion without interference from phenolic compounds, carbamate pesticides, triazine herbicides, and organophosphate pesticides that do not have the p-nitrophenyl substituent. The biosensor had excellent operational lifetime stability with no decrease in response for more than 40 repeated uses over a 12-h period when stored at room temperature, while its storage life was approximately 2 days when stored in the operating buffer at 4degreesC. (C) 2004 Wiley Periodicals, Inc.