The vesicular acetylcholine transporter (VAChT) has been identified and characterized based on the acquisition of high affinity vesamicol binding and proton-dependent, vesamicol-sensitive acetylcholine accumulation by a fibroblast cell line transfected with a clone from a rat pheochromocytoma cDNA library encoding this protein. The distribution of VAChT mRNA coincides with that reported for choline acetyltransferase (ChAT), the enzyme required for acetylcholine biosynthesis, in the peripheral and central cholinergic nervous systems. A human VAChT cDNA was used to localize the VAChT gene to chromosome 10q11.2, which is also the location of the ChAT gene. The entire sequence of the human VAChT cDNA is contained uninterrupted within the first intron of the ChAT gene locus. Transcription of VAChT and ChAT mRNA from the same or contiguous promoters within a single regulatory locus provides a previously undescribed genetic mechanism for coordinate regulation of two proteins whose expression is required to establish a mammalian neuronal phenotype.