Homogeneous cholinesterases inhibition by sodium fluoride was studied by many authors, using conventional techniques. Controversial results however were reported on the inhibition kinetics and mechanism. In this work electrochemical methods were applied to identify the type of sodium fluoride inhibition of the immobilized acety1cholinesterase taking advantage of the capabilities of the electrochemical biosensors. The acety1cholinesterase inhibition was evaluated by current measurement, at constant potential, of the mediated by K3[Fe(CN)61 and of the non-mediated oxidation of thiocholine iodide, produced by enzymatic acetylthiocholine iodide hydrolysis. Direct amperometric thiocholine detection was preferred for further studies, being more sensitive. The biosensor transducer response to thiocholine iodide was investigated by cyclic and hydrodynamic voltarnmetry. The reversibility and pH dependence (13112 = 1.08-0.06 pH) of thiocholine oxidation were demonstrated. The amperometric biosensor response to acetylthiocholine iodide obtained on a modified by acetylcholinesterase adsorption carbon electrode at +0.80V/Ag, AgC1 was characterised according to IUPAC recommendations. The enzyme reaction kinetic parameters: I..., K.PP, and K, were evaluated under kinetically controlled conditions, for various pH and temperature values. The reversibility ofthe inhibition was confirmed by dilution. Based on the kinetic analysis, the inhibition ofthe immobilized acetylcholinesterase by sodium fluoride was found to be of a competitive type. (c) 2008 Elsevier Ltd. All rights reserved.