A variety of approaches have been developed to localize neurons and neural elements in nervous system tissues that make and use acetylcholine (ACh) as a neurotransmitter. Choline acetyltransferase (ChAT) is the enzyme catalyzing the biosynthesis of ACh and is considered to be an excellent phenotypic marker for cholinergic neurons. We have surveyed the distribution of choline acetyltransferase (ChAT)-expressing neurons in the Drosophila nervous system detected by three different but complementary techniques. Immunocytochemistry, using anti-ChAT monoclonal antibodies results in identification of neuronal processes and a few types of cell somata that contain ChAT protein. In situ hybridization using cRNA probes to ChAT messenger RNA results in identification of cell bodies transcribing the ChAT gene. X-gal staining and/or beta-galactosidase immunocytochemistry of transformed animals carrying a fusion gene composed of the regulatory DNA from the ChAT gene controlling expression of a lacZ reporter has also been useful in identifying cholinergic neurons and neural elements. The combination of these three techniques has revealed that cholinergic neurons are widespread in both the peripheral and central nervous system of this model genetic organism at all but the earliest developmental stages. Expression of ChAT is detected in a variety of peripheral sensory neurons, and in the brain neurons associated with the visual and olfactory system, as well as in neurons with unknown functions in the cortices of brain and ganglia.