The Drosophila cholinergic locus is composed of two distinct genetic functions: choline acetyltransferase (ChAT; EC 2.3.1.6), the enzyme catalyzing biosynthesis of neurotransmitter acetylcholine (ACh), and the vesicular ACh transporter (VAChT), the synaptic vesicle membrane protein which pumps transmitter into vesicles. Both genes share a common first exon and the remainder of the VAChT gene contains a single coding exon residing entirely within the first intron of ChAT. RNase protection analysis indicates that all Drosophila VAChT specific transcripts contain the shared first exon and suggests common transcriptional control for ChAT and VAChT. Similar types of genomic organization have been evolutionarily conserved for cholinergic loci in nematodes and vertebrates, and may operate to ensure coordinate expression of these functionally related genes in the same cells. The relative levels of Drosophila ChAT and VAChT mRNA differ, however, in different tissues or in Cha mutants, indicating that independent regulation of ChAT and VAChT transcripts may occur post-transcriptionally. The predicted Drosophila VAChT protein is composed of 578 amino acids and contains 12 conserved putative transmembrane domains. Full-length VAChT cDNA is 7.2 kilobase long and has unusually long 5'- and 3'-untranslated regions (UTR). The 5'-UTR contains a GTG ChAT translational initiation codon along with three other potential ATG initiation codons. These features of the VAChT 5'-UTR region suggest that a ribosome scanning model may not be used for VAChT translation initiation.