Abstract A flow-injection procedure for the determination of paraoxon as an example of organophosphorus pesticides based on inhibition of acetylcholinesterase immobilized on the polymer carrier VA Epoxy Biosynth was studied. The detection was carried out spectrophotometrically by means of enzymatic hydrolysis of acetylthiocholine iodide and reaction of the thiocholine from the enzymatic reaction with 5,5′-dithiobis(2-nitrobenzoic acid). Under optimal conditions for an inhibition time of 30 min the calibration graph was linear from 0.05 to 0.5 μg l −1 ( r = 0.998, n = 5) with a relative standard deviation of 4.1% at 0.1 μg l −1. For an inhibition time of 3 min the calibration graph was linear from 2 to 20 μg l −1 with relative standard deviation ( n = 3) of 1.2% at 5 μg l −1. The inhibited enzyme was reactivated by 0.01 mol l −1 2-pyridinealdoxime methiodide. The recoveries from the samples to which paraoxon had been added (water, soil) were 104% and 94%, respectively.