The capacity of the high-affinity choline transporter (CHT) to import choline into presynaptic terminals is essential for acetylcholine synthesis. Ceramic-based microelectrodes, coated at recording sites with choline oxidase to detect extracellular choline concentration changes, were attached to multibarrel glass micropipettes and implanted into the rat frontoparietal cortex. Pressure ejections of hemicholinium-3 (HC-3), a selective CHT blocker, dose-dependently reduced the uptake rate of exogenous choline as well as that of choline generated in response to terminal depolarization. Following the removal of CHTs, choline signal recordings confirmed that the demonstration of potassium-induced choline signals and HC-3-induced decreases in choline clearance require the presence of cholinergic terminals. The results obtained from lesioned animals also confirmed the selectivity of the effects of HC-3 on choline clearance in intact animals. Residual cortical choline clearance correlated significantly with CHT-immunoreactivity in lesioned and intact animals. Finally, synaptosomal choline uptake assays were conducted under conditions reflecting in vivo basal extracellular choline concentrations. Results from these assays confirmed the capacity of CHTs measured in vivo and indicated that diffusion of substrate away from the electrode did not confound the in vivo findings. Collectively, these results indicate that increases in extracellular choline concentrations, irrespective of source, are rapidly cleared by CHTs.