Abstract: Messenger RNAs and the cognate gene(s) encoding choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) have been cloned from mammals and several other animal classes in the last decade. These have provided molecular tools for investigating acetylcholine synthesis and packaging into synaptic vesicles, the genesis of cholinergic vesicles, and the development and senescence of the cholinergic nervous system. VAChT and ChAT have been found to share a common gene locus and regulatory elements for gene transcription. The cholinergic gene locus represents a previously undiscovered type of neuronal transcriptional unit controlling chemically coded neurotransmission. In vitro assays for the transport function of VAChT have shed light on the bioenergetics of amine accumulation in secretory vesicles. Manipulation of VAChT expression in vivo has demonstrated unequivocally the primacy of vesicular exocytosis as the mode of transmitting quanta of acetylcholine at the neuromuscular junction, as in vivo manipulation of acetylcholinesterase levels has demonstrated the importance of acetylcholine metabolism in the regulation of complex functions such as cognition. Light and electron microscopic visualization of VAChT, complementing previous ChAT immunohistochemistry, has improved understanding of the genesis and function of the cholinergic vesicle, neuron, and synapse. These advances should accelerate the development of “cholinergic” pharmacological and gene therapeutic approaches to treatment of human diseases that are associated with cholinergic surfeit and insufficiency.