An electrochemical biosensor for organophosphorus (OP) pesticides trace level concentrations determination was developed and characterized. It integrates a hybrid biorecognition element consisting of immobilized Arthrobacter globiformis and free acetylcholinesterase (ACh) with a Clark type oxygen probe transducer. The bacteria convert the ACh-generated choline to betaine with oxygen consumption measured as a Clark probe current change. This change representing the sensor response correlates to the concentration of the OP pesticides inhibiting the Ach-catalyzed acetylcholine hydrolysis to choline. The conditions for maximal sensor response to choline were optimized according to the methodology of design of experiments. The analytical performances of the enzyme substrate determination in a wide concentration range (0.1 -20 mu mol dm(-3) of acetylcholine) and different ACh activities were established. It was demonstrated that the biosensor ensures reproducible, accurate and reliable chlorophos quantification reaching a limit of detection (LOD) of 1 nmol dm(-3) and a sensitivity of 0.0252 mu A/p(mol dm(-3)) under optimal experimental conditions. The biosensor response time is 200 sand the storage stability is t(L50) = 49 days for the bacterial membrane at ambient temperature. The device is reusable, the bacterial membrane being not affected by OR The biosensor was applied to chlorophos determination in contaminated milk. (C) 2008 Elsevier Ltd. All rights reserved.