Interstitial cells of Cajal (ICC) are interposed between enteric neurons and smooth muscle cells in gastrointestinal (GI) muscles. The specific relationships between these cells in the murine proximal colon were studied with conventional and immunoelectron microscopy and immunohistochemistry. Intramuscular interstitial cells (IC-IM) formed discrete networks within the circular muscle layer of the murine proximal colon. Nerve trunks ran in close association with IC-IM and individual nerve trunks came into close contact with multiple IC-IM. Conventional electron microscopy revealed very close (< or = 20 nm) associations between nerve fibers and IC-IM. Processes of IC-IM also formed close contacts with neighboring smooth muscle cells. At the points of close association between neurons and IC-IM, areas of membrane densification in both pre- and postjunctional cells were present, suggesting specialized contacts or synaptic-like structures. Similar points of contact between neurons and smooth muscle cells were extremely rare. Immunoelectron microscopy demonstrated that IC-IM formed close associations with neurons containing nitric oxide synthase-like immunoreactivity (NOS-LI) or vesicular acetylcholine transporter-like immunoreactivity (vAChT-LI), suggesting innervation by both inhibitory and excitatory motor neurons. IC-IM were also labeled with anti-NOS antibodies. These observations suggest that IC-IM are an integral part of the neuromuscular junction in the colon. These cells may be the primary site of innervation, and neural regulation of the musculature may occur via IC-IM.