Acetylcholine is known to be a neurotransmitter in crustacean central nervous systems, but the numbers and distribution of cholinergic neurons in the segmental ganglia have not been described. To begin a census of cholinergic neurons in these ganglia, we used a histochemical assay for acetylcholinesterase to map neurons that contained this enzyme in the six abdominal ganglia of crayfish. In each abdominal ganglion, about 47 cell bodies were stained. The distributions of these stained cells in individual ganglia were similar, and the numbers were not significantly different. None of these stained cell bodies could be identified from their structures or locations as previously identified motor neurons or sensory neurons with central cell bodies. The process of one unpaired midline neuron that occurred only in the first three abdominal ganglia divided to send a pair of axons anteriorly into both halves of the connective. The central projections of afferent axons from many peripheral sensory neurons stained clearly as they entered each ganglion. Terminals of these axons were heavily stained in the horseshoe neuropil and the lateral neuropils. We labeled both gamma-aminobutyric acid (GABA) and acetylcholinesterase in individual ganglia. Only a few neurons in each ganglion were double-labeled. The unpaired midline neurons in the three anterior ganglia that stained for acetylcholinesterase did not show GABA-like immunoreactivity, but cells with similar shapes did label with the GABA antiserum. Acetylcholinesterase is not a definitive marker of cholinergic neurons, but its presence is often associated with the cholinergic phenotype. These stained cells should be considered as putative cholinergic neurons.