Functional high- and low-affinity choline transport processes from rat cortical plasma membranes were reconstituted in phosphatidylcholine bilayer liposomes. The high-affinity choline transporter demonstrated a pharmacological profile and ion dependency that were identical to those of intact synaptosomes. This preparation may be used to further characterize choline transport and, with appropriate supplementation, to investigate the release of acetylcholine in the absence of synaptic vesicles.