Lipophilic, alkylated cyclodextrins (CDs) are aromatic hosts which are excellent ionophores for reversibly binding size-matched, charge-diffuse cations. The application of lipophilic beta-CDs as ionophores for sensing acetylcholine chloride using a potentiometric ISE and an amperometric biosensor is described. Potentiometric ISEs, using 2,6-didodecyl-beta-CD as ionophore, showed a Nernstian response with a limit of detection -log[C] = 5.0 and selectivity coefficient -logKijPot = 4.2 (in serum levels of Na+, K+ and Ca2+). A Nernstian response is maintained in the presence of bovine serum albumin for a PVC- and a polyurethane-based electroactive membrane on initial contact. However, on prolonged contact, the polyurethane-based membranes showed a lower shift in Einitial0 (the bias potential on initial contact with analyte solution) than PVC. Amperometric biosensors were assembled by modifying screen-printed electrodes with a ferrocenyl charge shuttle, enzymes (horseradish peroxidase, choline oxidase and/or acetylcholine esterase) and a thin film comprising a polyurethane matrix, 2,3,6-triethyl-beta-CD, a plasticizer and a large anionic salt. The resulting sensor, which was capable of detecting subpicomolar levels of acetylcholine, was highly specific and was stable to storage in air and in solution. Interference from endogenous electroactive compounds was minimal.