Recent studies have demonstrated that intramuscular interstitial cells of Cajal (ICC) are preferential targets for neurotransmission in the stomach. ICC in the small intestine and colon also form tight, synaptic-like contacts with terminals of enteric motor neurons, but little is known about the role of these cells in neurotransmission. ICC at the deep muscular plexus (ICC-DMP) of the small intestine express neurokinin 1 receptors (NK1R) and internalize these receptors in response to exogenous substance P. We used NK1R internalization as an assay of functional innervation of ICC-DMP in the murine small intestine. Under basal conditions NK1R-like immunoreactivity (NK1R-LI) was mainly observed in ICC-DMP (519 cells counted, 100% were positive) and myenteric neurons. ICC-DMP were closely apposed to substance P-containing nerve fibers. Of 338 ICC-DMP examined, 65% were closely associated with at least 1 substance P-positive nerve fiber, 32% were associated with at least 2, 2% were associated with more than 2 nerve fibers and 1% with none. After electrical field stimulation (EFS, 10 Hz; 1 minute) NK1R-LI was internalized in more than 80% of ICC-DMP, as compared to 10% of cells before EFS. Internalization of NK1R was not observed in myenteric ICC or smooth muscle cells in response to nerve stimulation. Internalization of NK1R-LI was blocked by the specific NK1 receptor antagonist WIN 62577 (1 µM) and by tetrodotoxin (0.3 µM), suggesting that internalization resulted from stimulation of receptors with neurally-released neurokinins. These data suggest that ICC-DMP are primary targets for neurokinins released from enteric motor neurons in the intestine.