Background and Aims: Choline acetyltransferase, an enzyme involved in the synthesis of acetylcholine, is a marker of cholinergic neurons. In this study, the distribution of choline acetyltransferase immunoreactivity in human intestine is described. Methods: Frozen-section and whole-mount preparations of human small and large bowels were made and labeled with antiserum to choline acetyltransferase. Double labeling with antiserum to neuron-specific enolase enabled the proportion of all neurons that were immunoreactive for choline acetyltransferase to be determined. Results: Nerve fibers, immunoreactive for choline acetyltransferase, were frequent in the circular and longitudinal muscle layers and were widespread in the myenteric and submucous plexuses, but none was observed in the mucosa. Myenteric neurons, immunoreactive for choline acetyltransferase, showed various morphologies, the most common being unipolar and having an irregular outline with several short, lamellar processes. Sixty-four percent of all myenteric neurons were immunoreactive for choline acetyltransferase. Cholinergic submucous neurons were homogeneous in appearance with oval, smooth cell bodies and filamentous dendrites and accounted for 53% of all submucous neurons. A number of cells resembling enteroendocrine cells in the epithelium of the small and large bowels had intense choline acetyltransferase immunoreactivity. Conclusions: The majority of neurons in human small and large intestines are cholinergic.