Abstract An accurate method to count human enteric neurons is essential to develop a comprehensive account of the classes of nerve cells responsible for gut function and dysfunction. The majority of cells in the enteric nervous system utilize acetyl choline, or nitric oxide, or a combination of these, as neurotransmitters. Antisera raised against the RNA-binding protein Hu, were used to identify nerve cell bodies in whole mounts of the myenteric plexus of human colon, and then were utilized to analyse cells immunoreactive for combinations of choline acetyltransferase and nitric oxide synthase. Antisera to Hu provided a reliable means to count apparently all enteric nerve cell bodies, revealing 10% more cell bodies than labelling with neuron specific enolase, and no labelling of glial cells as revealed by S100. ChAT+/NOS− neurons accounted for 48% (±3%) of myenteric neurons and ChAT−/NOS+ neurons accounted for 43% (±2.5%). ChAT+/NOS+ neurons comprised 4% (±0.5) of the total number of neurons, and a novel class of small ChAT−/NOS− neurons, making up 5% (±0.9%) of all cells, was described for the first time.