c-Kit-immunopositive interstitial cells (equivalent to interstitial cells of Cajal: ICC) are currently recognised to play a crucial role in gastrointestinal (GI) pacemaking (Takaki, 2003). One of the characteristic features of GI spontaneous rhythmicity is Ca²⁺ dependence (Tomita, 1981). Also, it has been recently suggested that activation of a Cl^– conductance is responsible for pacemaker potentials (Dickens et al. 1999; Huizinga et al. 2002). It is therefore speculated that cytosolic Ca²⁺ ([Ca²⁺]_i) oscillations are the primary mechanism in GI pacemaker cells, i.e. Ca²⁺-activated Cl^– channels and/or other Ca²⁺-activated channels (Walker et al. 2002) are periodically activated by [Ca²⁺]_i oscillations in ICCs. Cell cluster preparations enzymatically isolated from GI tract contain smooth muscle, enteric neurones and ICCs, and are therefore considered to consist of the essential minimum cell members necessary to investigate mechanisms underlying GI motility and pacemaker function. In this communication, we show properties of [Ca²⁺]_i oscillations in ICCs, using cell cluster preparations from mouse stomach and small intestine after incubation in culture medium for several days. The mice were humanely killed. Dihydropyridine Ca²⁺ antagonists (1 μM nifedipine) were used to monitor Ca²⁺ signals from ICCs. Pacemaker [Ca²⁺]_i oscillations in both stomach and small intestine were affected by drugs interacting with ryanodine receptors (RyR) (e.g. ryanodine 1-10 μM), inositol 1,4,5-trisphosphate (InsP₃) receptors (e.g. 10 μM xestospongin C) and TRP homologues (e.g. 40 μM SK&F96365). It is speculated that coordinating actions of different types of intracellular Ca²⁺ release channels produce [Ca²⁺]_i oscillations in ICCs, supported by Ca²⁺ influx via TRP family channels. RT-PCR examinations detected the cDNA of the molecular component of these channels in isolated ICCs identified by c-Kit-immunoreactivity. Involvement of RyR was also suggested in spontaneous rhythmicity of gut-like organ formed from mouse embryonic stem cells. We further investigated the effects of K⁺ channel openers (e.g. 1-10 μM cromakalim) and sulphonylurea compounds (1 μM glibenclamide). The results suggest that sulphonylurea receptors differentially modulate pacemaker [Ca²⁺]_i oscillations in ICCs and smooth muscle contractility.