The distribution of the high-affinity choline transporter (CHT) was determined in the human and macaque monkey spinal cord using in situ hybridization histochemistry and immunohistochemistry. Signals for CHT mRNA were observed in somatic motor neurons, sympathetic preganglionic neurons, and neurons in the medial part of lamina VII. The mRNA for CHT was co-localized in single neurons with the mRNAs for vesicular acetylcholine transporter and cholineacetyltransferase. These same cholinergic neuronal groups were labeled by immunohistochemistry for human CHT. Of somatic motor neurons, smaller cell bodies of gamma-motor neurons were labeled very intensely, whereas larger cell bodies of alpha-motor neurons showed various degrees of labeling from weak to moderately intense. Human CHT is thus a novel cholinergic marker, which not only labels cholinergic neurons, but also reveals their heterogeneity.