Abstract We investigated whether substance P modulates pacemaker currents generated in cultured interstitial cells of Cajal of murine small intestine using whole cell patch-clamp techniques at 30 °C. Interstitial cells of Cajal generated spontaneous inward currents (pacemaker currents) at a holding potential of −70 mV. Tetrodotoxin, nifedipine, tetraethylammonium, 4-aminopyridine, or glibenclamide did not change the frequency and amplitude of pacemaker currents. However, divalent cations (Ni 2+, Mn 2+, Cd 2+, and Co 2+), nonselective cationic channel blockers (gadolinium and flufenamic acid), and a reduction of external Na + from normal to 1 mM inhibited pacemaker currents indicating that nonselective cation channels are involved in their generation. Substance P depolarized the membrane potential in current clamp mode and produced tonic inward pacemaker currents with reduced frequency and amplitude in voltage clamp mode. [ d-Arg 1, d-Trp 7,9, Leu 11] substance P, a tachykinin NK 1 receptor antagonist, blocked these substance P-induced responses. Furthermore, [Sar 9, Met(O 2) 11] substance P, a specific tachykinin NK 1 receptor agonist, depolarized the membrane and tonic inward currents mimicked those of substance P. Substance P continued to produce tonic inward currents in external Ca 2+-free solution or in the presence of chelerythrine, a protein kinase C inhibitor. However, substance P-induced tonic inward currents were blocked by thapsigargin, a Ca 2+-ATPase inhibitor in the endoplasmic reticulum or by an external 1 mM Na + solution. Our results demonstrate that substance P may modulate intestinal motility by acting on the interstitial cells of Cajal by activating nonselective cation channels via the release of intracellular Ca 2+ induced by tachykinin NK 1 receptor stimulation.