Cytochrome P450-3A4 (CYP3A4) is a monooxygenase enzyme that plays a major role in the detoxification of bioactive compounds and hydrophobic xenobiotics ( e. g. medicines, drugs, environmental pollutants, food supplements and steroids). Physiologically the monooxygenation reactions of this class II, microsomal, b-type heme enzyme, usually requires cytochrome P450 reductase, NADPH. A novel CYP3A4 biosensor system that essentially simplified the enzymatic redox processes by allowing electron transfer between the electrode and the enzyme redox centre to occur, without any need for the physiological redox partners, was developed for the detection of 2,4-dichlorophenol (2,4-DCP), a priority environmental pollutant and an endocrine disruptor. The biosensor, GC/Naf-Co(Sep)(3+)/CYP3A4/Naf, was constructed by encapsulating CYP3A4 in a Nafion-cobalt (III) sepulchrate (Naf-Co(Sep)(3+)) composite film on a glassy carbon (GC) electrode. The responses of the biosensor to 2,4-dichlorophenol, erythromycin (CYP3A4 native substrate) and ketoconazole (CYP 3A4 natural inhibitor) were studied by cyclic and square wave voltammetric techniques. The detection limit (DL) of the biosensor for 2,4-dichlorophenol was 0.043 mu gL(-1), which is by an order of magnitude lower than the EU limit (0.3 mu gL(-1)) for any pesticide compound in ground water. The biosensor's DL is lower than the U. S. Environmental Protection Agency's drinking water equivalent level (DWEL) value for 2,4-DCP, which is 2 mu gL(-1). (C) 2008 Elsevier Ltd. All rights reserved.