We use a transgenic mouse model system to elucidate the regulatory regions within the human cholinergic gene locus responsible for vesicular acetylcholine transporter gene expression in vivo. In this report we characterized two transgenes for their ability to confer cholinergic-specific expression of the encoded vesicular acetylcholine transporter. An 11.2 kb transgene (named hV11.2) that spanned from about 5 kb upstream of the start of vesicular acetylcholine transporter translation down to the first choline acetyltransferase coding exon gave expression in the somatomotor neurons and a subpopulation of cholinergic neurons in the medial habenular nucleus. The second transgene (named hV6.7), a 5-prime truncated version of hV11.2 that was devoid of 4.5 kb of gene-regulatory sequences completely lacked vesicular acetylcholine transporter expression in vivo. Our data indicate that vesicular acetylcholine transporter expression in somatomotor neurons and in the medial habenular nucleus is uniquely specified within the cholinergic gene locus, and separable from cholinergic expression elsewhere. The identification of these two subdivisions of the cholinergic nervous system suggests that other cholinergic neurons in the CNS and PNS are similarly regulated by additional discrete domains within the cholinergic gene locus.