High-affinity choline transport (HAChT), the rate-limiting and regulatory step in acetylcholine (ACh) synthesis, is selectively localized to cholinergic neurons. Hemicholinium-3 (HC3), a potent and selective inhibitor of HAChT, has been used as a specific radioligand to quantify HAChT sites in membrane binding and autoradiographic studies. Because both HAChT velocity and [3H]HC3 binding change as in vivo activity of cholinergic neurons is altered, these markers are also useful measures of cholinergic neuronal activity. Evidence that [3H]HC3 is a specific ligand for HAChT sites on cholinergic terminals is reviewed. The ion requirements of HAChT and [3H]HC3 binding indicate that sodium and chloride are required for recognition of both choline and [3H]HC3. A common recognition site is also indicated by the close correspondence of the potency of HC3 and choline analogues for inhibiting both HAChT and [3H]HC3 binding. The parallel regional distributions of both markers in adult brain, during development and after specific lesions, all indicate specific cholinergic localization. The close association of HAChT and [3H]HC3 binding sites is also supported by parallel regulatory changes occurring after in vivo drug treatments and in vitro depolarization. Overall, the data indicate a close association between HAChT and [3H]HC3 binding and are consistent with the sites being identical. Methodologic considerations in using [3H]HC3 as a ligand and considerations in interpretation of results are also discussed.