Although many studies show that thromboxane A2 (TXA2) has the action of gastrointestinal (GI) motility using GI muscle cells and tissue, there are no reports on the effects of TXA2 on interstitial cells of Cajal (ICC) that function as pacemaker cells in GI tract. So, we studied the modulation of pacemaker activities by TXA2 in ICC with whole cell patch-clamp technique. Externally applied TXA2 (5ՌM) produced membrane depolarization in current-clamp mode and increased tonic inward pacemaker currents in voltage-clamp mode. The tonic inward currents by TXA2 were inhibited by intracellular application of GDP-Ղ-S. The pretreatment of ICC with Ca2+ free solution and thapsigargin, a Ca2+-ATPase inhibitor in endoplasmic reticulum, abolished the generation of pacemaker currents and suppressed the TXA2-induced tonic inward currents. However, chelerythrine or calphostin C, protein kinase C inhibitors, did not block the TXA2-induced effects on pacemaker currents. These results suggest that TXA2 can regulate intestinal motility through the modulation of ICC pacemaker activities. This modulation of pacemaker activities by TXA2 may occur by the activation of G protein and PKC independent pathway via extra and intracellular Ca2+ modulation.