Biosensor for measuring acetylcholine with application to cholinergic synaptic vesicles. A highly sensitive and selective acetylcholine biosensor was developed by incorporating acetylcholinesterase and choline oxidase into a lipid bilayer. The sensor was used to detect acetylcholine released from cholinergic synaptic vesicles. The acetylcholine biosensor demonstrated a linear response over a wide concentration range (0.5-800 microM). This work represents the first time a choline-oxidase-based biosensor has been developed that can measure low levels of acetylcholine release with accuracy, sensitivity and good linear dynamic range. The response of the sensor was dependent on the ratio of choline oxidase to acetylcholinesterase, as well as the ionic conditions (pH, sodium ion concentration). The sensor displayed an extremely fast response time (<1 s) and was responsive for at least 2 h. The choline-oxidase-based biosensor described here is expected to provide a powerful tool for the study of acetylcholine release from cholinergic synaptic vesicles.