It is well established that acetylcholine is a neurotransmitter at several distinct sites in the mammalian enteric nervous system. However, identification of the cholinergic neurons has not been possible due to an inability to selectively label enteric cholinergic neurons. In the present study an immunohistochemical method has been developed to localize choline acetyltransferase, the synthetic enzyme for acetylcholine, in order that cholinergic neurons can be visualized. The morphology, neurochemical coding and projections of cholinergic neurons in the guinea-pig small intestine were determined using double-labelling immunohistochemistry. These experiments have revealed that many myenteric neurons are cholinergic and that they can be distinguished by their specific combinations of immunoreactivity for neurochemicals such as calretinin, neurofilament protein triplet, substance P, enkephalin, somatostatin, 5-hydroxytryptamine, vasoactive intestinal peptide and calbindin. On the basis of their previously described projections, functional roles could be attributed to each of these populations. The identified cholinergic neurons are: motorneurons to the longitudinal muscle (choline acetyltransferase/calretinin); motorneurons to the circular muscle (choline acetyltransferase/neurofilament triplet protein/substance P, choline acetyltransferase/substance P and choline acetyltransferase alone); orally directed interneurons in the myenteric plexus (choline acetyltransferase/calretinin/enkephalin); anally directed interneurons in the myenteric plexus (choline acetyltransferase/somatostatin, choline acetyltransferase/5-hydroxytryptamine, choline acetyltransferase/vasoactive intestinal peptide); secretomotor neurons to the mucosa (choline acetyltransferase/somatostatin); and sensory neurons mediating myenteric reflexes (choline acetyltransferase/calbindin). This information provides a unique opportunity to identify functionally distinct populations of cholinergic neurons and will be of value in the interpretation of physiological and pharmacological studies of enteric neuronal circuitry.