Cholinergic neurons have been revealed in the enteric nervous system by functional and biochemical studies but not by antibodies that provide excellent localisation of the synthesising enzyme, choline acetyltransferase (ChAT), in the central nervous system. In order to determine whether a newly described peripheral form of ChAT (pChAT) is a ChAT enzyme of enteric neurons, we have compared pChAT distribution with that of the common form of ChAT, cChAT, by quantitative analysis of the co-localisation of pChAT and cChAT with other neurochemical markers in enteric neurons of the guinea-pig ileum. We found classes of neuron with strong pChAT immunoreactivity (IR) and others with strong cChAT-IR. In myenteric ganglia, strong pChAT-IR was in calbindin-positive intrinsic primary afferent neurons (IPANs), whereas cChAT-IR of these neurons was weak. Calretinin neurons were immunoreactive for cChAT, but not pChAT. Only 4% of nitric oxide synthase (NOS) neurons (possibly interneurons) were pChAT-immunoreactive, similar to observations with cChAT. NOS-immunoreactive inhibitory motor neurons stained with neither cChAT nor pChAT antisera. In the submucosal ganglia, pChAT-IR was strongly expressed in IPANs (identified by cytoplasmic staining for the neuronal nuclear marker, NeuN) and in neuropeptide Y (NPY)-immunoreactive secretomotor neurons, but not in calretinin-immunoreactive neurons. cChAT-IR occurred weakly in submucosal IPANs and also labelled NPY- and calretinin-immunoreactive neurons. Submucosal vasoactive-intestinal-peptide-immunoreactive neurons (non-cholinergic secretomotor neurons) were not reactive for either form of ChAT.