(A) Shows segments of plasma membrane (PM) and endoplasmic reticulum membrane (ER) that form PM-ER junctions and microdomains. At least three types of voltage-dependent Ca2+ channels (VDCC) are expressed in ICC-SM, CaV1.2, CaV1.3, and CaV3.2. These conductances, with voltage-dependent activation and inactivation properties spanning a broad range of negative potentials, insure maintenance of pacemaker activity under conditions of hyperpolarization or depolarization in ICC-SM. Pacemaker activity (1. Initial events) in ICC-SM could be due to spontaneous release of Ca2+ from stores in the ER and utilize either IP3R or RYR receptors or both (Ca2+ sparks and puffs). However, our data cannot rule out the possibility that transient openings of voltage-dependent Ca2+ channels (sparklets) and amplification of Ca2+ in microdomains by CICR constitute the initial events of pacemaker activity. In this case, Ca2+ release from stores is not the primary pacemaker event but a secondary response to Ca2+ entry. Inhibition of Ca2+ release from stores would lead to reduced probability of CICR and decrease the frequency of CTCs. Our hypothesis is that Ca2+ entry and/or release from stores activates Ca2+-dependent Cl- current due to ANO1 channels in the plasma membrane. Active propagation between cells in interstitial cells of Cajal (ICC) networks (Phase 2) was inhibited by blocking voltage-dependent Ca2+ channels. Active propagation may also require or depend upon amplification of Ca2+ in microdomains by CICR. The duration of Ca2+ entry is likely to be brief due to voltage-dependent inactivation of L- and T-type Ca2+ channels. The duration of CTCs appears to be enhanced by CICR (Phase 3). Our data show that the duration of CTCs is reduced by several manipulations known to inhibit Ca2+ release from stores. In Phase four store reloading may occur by multiple mechanisms and may include: (i) transient Ca2+ entry via sparklets, (ii) activation of SOCE via STIM/ORAI interactions, and (iii) the increase in Ca2+ entry that occurs via depolarization and activation of Ca2+ entry at the onset of each CTC. (B) A novel hypothesis emerges from this study suggesting that the pacemaker mechanism in non-voltage-clamped cells includes a cyclical, positive-feedback phenomenon that may be responsible for initiation of CTCs and relies on: (i) Ca2+ entry through voltage-dependent Ca2+ channels. Openings of clusters of these channels would generate sparklets; (ii) Ca2+ entry initiates CICR which amplifies [Ca2+]i within microdomains; (iii) the rise in [Ca2+]i activates ANO1 channels in the PM causing depolarization; (iv) depolarization enhances the open probability of voltage-dependent Ca2+ channels, increasing Ca2+ entry. This cycle creates positive feedback for Ca2+ entry, clustering of localized Ca2+ transients due to Ca2+ entry during the first 350–450 ms of CTCs and development of slow wave depolarizations in ICC-SM.