Under many physiological circumstances, Na+- and Cl--dependent, hemicholinium-3 (HC-3)-sensitive, high-affinity choline uptake (HACU) in cholinergic neurons is thought to be rate-limiting in the biosynthesis of acetylcholine (ACh). Based on sequence information provided by the Human Genome Project and the recently reported rat CHT1 (rCHT1), me cloned a human CHT cDNA from spinal cord. The hCHT cDNA encodes a protein of 580 amino acids having 93% identity to rCHT1 and 51% identity to the Caenorhabditis elegans homolog CHO-1, and is distantly related to members of the Na+-coupled glucose transporter (SGLT) gene family of Naf-coupled glucose (SGLT), nucleoside and iodide transporters. Northern blot analysis reveals the expression of a similar to 5 kb transcript in human brain regions rich in cholinergic neurons including the putamen, spinal cord, and medulla. Expression of hCHT cDNA in COS-7 cells results in saturable, Na+/Cl--dependent choline uptake (K-m = 1.2 mu M) in membrane vesicles and [H-3] HC-3 binding (K-d = 4 nM) in membrane fractions, consistent with characteristics reported in mammalian cholinergic neurons. Using radiation hybrid mapping techniques, me localized the hCHT gene to human chromosome 2q12. These studies elucidate the primary structure and chromosomal localization of hCHT and provide a basis for mechanistic analysis of HACU regulation and an investigation of the role of hCHT in disease states.