An electrochemical sensor was applied for investigating the immobilized rat brain acetylcholinesterase inhibition by chlorofos. Two alternative routes were explored as response-generating reactions: (i) direct electrochemical oxidation of thiocholine produced upon acetylthiocholine enzymatic hydrolysis and (ii) reduction of the produced thiocholine with hexacyanoferrate (III), followed by hexacyanoferrate (II) electrochemical detection. The advantages of the direct way are simplicity and higher sensitivity compared to the indirect one, which however avoids the interferences because of the lower potential applied. Enzyme inhibition was identified as competitive, the K-M(app) increasing from 1.31 to 1.43 mmol L-1 with chlorofos concentration in the range 0.2-1.0 mmol L-1 and the maximal rate of the enzyme reaction remaining constant (I-max = 579.30 +/- 5.71 mu A) in the presence of chlorofos. The inhibition constant was calculated using the Dixon method (K-I = 10.07 mmol L-1). The suppression of the acetylcholinesterase activity by the inhibitor, expressed as current decrease at a constant substrate concentration, was exploited for chlorofos quantification optimized by the design of experiments methodology. Optimal response was obtained for an acetylthiocholine concentration of 0.2 mmol L-1, at 26 degrees C and pH 7. (C) 2009 Elsevier Ltd. All rights reserved.