Amperometric acetylcholinesterase biosensors have been developed for quantification of the pesticides carbofuran, carbaryl, methylparaoxon, and dichlorvos in phosphate buffer containing 5% acetonitrile. Three different biosensors were built using three different acetylcholinesterase (AChE) enzymes-AChE from electric eel, and genetically engineered (B394) and wild-type (B1) AChE from Drosophila melanogaster. Enzymes were immobilized on cobalt(II) phthalocyanine-modified electrodes by entrapment in a photocrosslinkable polymer (PVA-AWP). Each biosensor was tested against the four pesticides. Good operational stability, immobilisation reproducibility, and storage stability were obtained for each biosensor. The best detection limits were obtained with the B394 enzyme for dichlorvos and methylparaoxon (9.6 x 10(-11) and 2.7 x 10(-9) mol L(-1), respectively), the B1 enzyme for carbofuran (4.5 x 10(-9) mol L(-1)), and both the B1 enzyme and the AChE from electric eel for carbaryl (1.6 x 10(-7) mol L(-1)). Finally, the biosensors were used for the direct detection of the pesticides in spiked apple samples.