Abstract: To determine paraoxon (I), the sample solution (60 µL) was injected into a carrier stream of 50 mM potassium phosphate buffer of pH 8 (1 ml/min) flowing through a PTFE manifold (50 µm i.d.). A solution of 7 mM acetylthiocholine iodide (60 µL) was injected into the centre of the I plug 10 cm downstream of the first injection point and the stream was passed through a glass column (2 cm x 2.5 mm i.d.) of acetylcholinesterase immobilized on controlled-pore-glass beads. The presence of I in the column inhibited the catalytic activity of the enzyme in the hydrolysis of acetylthiocholine iodide to thiocholine determined spectrophotometrically according to Quintero et al. (Talanta, 1991, 38, 1273) after mixing the column effluent with 1 mM 5,5'-dithiobis(2-nitrobenzoic acid). The total uninhibited catalytic activity of the acetylcholinesterase was determined similarly by injecting acetylthiocholine iodide after reactivation of the enzyme by pyridine-2-aldoxime methochloride. Calibration graphs were linear up to 0.8 mM I and the detection limit was 50 µM-I. The RSD (n = 5) was 5% for 0.6 mM I. A procedure for determining up to 0.6 mM carbamoylcholine was also described.