A simple, accurate, precise and sensitive potentiometric assay for the determination of acetycholine (Ach) in pure and biological samples is described. The method is based on the construction of an Ach sensor using carbon paste containing 1-10% (w/w) 2,3,6-trimethyl o-beta-cyclodextrin doped as an ionophore with tricresyl phosphate (TCP) (a plasticizer) and potassium tetrakis-p-chlorophenyl borate (TKB). The increase in the potentiometric response of the carbon paste electrode CPE toward Ach is characterized by a slope of 57.4 +/- 0.5 mV/decade in the range of 10(3)-10(-5) M Ach solution. The detection limit and correlation factors are calculated. The optimization of various experimental conditions is described. A successful application of the new CPE to determine Ach in the homogenates. of cotton leaf worm "Spodoptera littoralis" is presented. (C) 2004 Elsevier B.V. All rights reserved.