Background Aims: The interstitial cells of Cajal (ICC) are pacemaker cells in gastrointestinal tract and generate an electrical rhythm in gastrointestinal muscles. We investigated the possibility that PGE2 might affect the electrical properties of cultured ICC by activating ATP-dependent K+ channels and, the EP receptor subtypes and the subunits of ATP-dependent K+ channels involved in these activities were identified. In addition, the regulation of intracellular Ca2+ (〔Ca2+〕i) mobilization may be involved the action of PGE2 on ICC. Results: Treatments of ICC with PGE2 inhibited electrical pacemaker activities in the same manner as pinacidil, an ATP-dependent K+ channel opener and PGE2 had only a dose-dependent effect. Using RT-PCR technique, we found that ATP-dependent K+ channels exist in ICC and that these are composed of Kir 6.2 and SUR 2B subunits. To characterize the specific membrane EP receptor subtypes in ICC, EP receptor agonists and RT-PCR were used: Butaprost (an EP2 receptor agonist) showed the actions on pacemaker currents in the same manner as PGE2. However sulprostone (a mixed EP1 and EP3 agonist) had no effects. In addition, RT-PCR results indicated the presence of the EP2 receptor in ICC. To investigate cAMP involvement in the effects of PGE2 on ICCs, SQ-22536 (an inhibitor of adenylate cyclase) and cAMP assays were used. SQ-22536 did not affect the effect of PGE2 on pacemaker currents, and PGE2 did not stimulate cAMP production. Also, we found PGE2 inhibited the spontaneous ［Ca2+］i oscillations in cultured ICC. Conclusions: These observations indicate that PGE2 alters pacemaker currents by activating the ATP-dependent K+ channels comprised of Kir 6.2-SUR 2B in ICC and this action of PGE2 are through EP2 receptor subtype and also the activation of ATR-dependent K+ channels involves intracellular Ca2+ mobilization.