The development and application of a method for the determination of pesticides and heavy metals by means of a biosensor system are described. Chemometrics, especially experimental design and simplex optimization proved to be a very valuable tool for this task. The system consists of three pH electrodes whose ion sensitive areas are connected with a membrane on which one enzyme is immobilized at a time. As biological components, acetylcholinesterase, alkaline and acid phosphatase were used; the latter two were employed for the first time in such a way. By potentiometrically detecting the pH shifts of the typical reactions of the immobilized enzymes with the corresponding substrates before and after an incubation time, the relative inhibition of eleven pesticides was determined by forming the ratio of both the potential differences. The combination of the inhibition results of three enzyme electrodes enables to get more information, both additional and excluding, about these pollutants than with only one enzyme electrode. In this way, a separation scheme of the used substances to be detected could be derived.