The high-affinity, hemicholinium-3-sensitive choline transporter (CHT) is responsible for acquisition of choline as a precursor in the synthesis of acetylcholine (ACh). Previous autoradiographic and functional studies suggest a localization of CHT to cholinergic neurons and terminals. We have reported the cloning and characterization of human and mouse choline transporters (Apparsundaram et al. Biochem. Biophy. Res. Commun. 276:862-867, 2001 and SFN abstract 26:306.5, 2000). We have developed polyclonal and monoclonal antibodies to permit the localization of CHT protein in situ. Western analysis with affinity purified polyclonal antibodies identifies a single, 70 kDa species in mouse brain that carries N-glycosylation as revealed by PNGAse F digestion studies. Immunocytochemical studies using these antibodies along with probes for choline acetyltransferase (ChAT), vesicular ACh transporter (VAChT) or synaptophysin demonstrate labeling of cholinergic neurons in the striatum, basal forebrain and brainstem, a...