In the digestive system, substance P is an excitatory transmitter to muscle, a putative excitatory neuro-neuronal transmitter, a vasodilator, and a mediator in inflammatory processes. Many of the biological effects of substance P are mediated by a high-affinity interaction with the tachykinin receptor neurokinin-1. The aim of the present study was to identify the sites of expression of this receptor in the rat stomach and intestine by immunohistochemistry with a polyclonal antiserum raised to the intracellular C-terminal portion of the rat neurokinin-1 receptor. Neurokinin-1 receptor immunoreactivity is present in a large population of enteric neurons. The relative density of these neurons along the gut is colon > ileum >> stomach. In the intestine, stained neurons have a smooth cell body with processes that can be followed within and between plexuses, and make close approaches to other neuronal cells, but do not appear to project outside the plexuses, suggesting that they are interneurons. In the stomach, neurokinin-1 receptor-immunoreactive neurons are infrequent and have a poorly defined and irregular shape. Neurokinin-1 receptor immunoreactivity is also localized to numerous non-neuronal cells in the inner portion of the circular muscle layer of the small intestine, which have the appearance of small dark smooth muscle cells or interstitial cells of Cajal. These cells are postulated to form a "stretch-sensitive" system with the deep muscular plexus and thus constitute an important site of regulation of muscle activity. Double labeling immunofluorescence was used to simultaneously localize neurokinin-1 receptor and substance P/tachykinin immunoreactivities. These experiments demonstrate that in the enteric plexuses, substance P/tachykinin-immunoreactive varicose fibers encircle the cell bodies of most neurokinin-1 receptor-containing neurons, and in the inner portion of the circular muscle layer of the small intestine they lie close to neurokinin-1 receptor-immunoreactive non-neuronal cells. In addition, some enteric neurons express both neurokinin-1 receptor and substance P/tachykinin immunoreactivities. The present study provides strong evidence that the neurokinin-1 receptor is the tachykinin receptor mediating the actions of substance P on enteric neurons and smooth muscle.