Parasympathetic control of gastrointestinal smooth muscles is mediated through muscarinic receptors (M₂ and M₃ subtypes), activation of which by acetylcholine causes depolarization of the smooth muscle cell (SMC) membrane via cationic channel opening. Membrane depolarization increases the frequency of action potentials (APs) and thereby facilitates Ca²⁺ entry through voltage-gated Ca²⁺ channels (VGCCs). Activation of M₂ receptors primarily gates the cationic channels via the α- GTP subunit of G_o protein, whereas activation of M₃ receptors is coupled via G_q/11 protein to stimulation of phospholipase C-β, which hydrolyses phosphatidylinositol-4,5-bisphosphate to diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃). While DAG has no effect on muscarinic cationic channels, Ca²⁺ mobilized from intracellular stores in response to IP₃ and Ca²⁺ entry through VGCCs potentiates muscarinic cationic current (mI_cat). The experiments were conducted on single SMCs freshly isolated from the longitudinal layer of the guinea-pig ileum. The animals were humanely killed. Using fluorescence confocal imaging we analysed the spatio-temporal profile of [Ca²⁺]_i changes following stimulation of muscarinic receptors with carbachol (CCh) and related the spatial pattern of CCh-induced [Ca²⁺]_i transients to the spatial distribution of the intracellular Ca²⁺ stores, IP₃ receptors (IP₃Rs) and ryanodine receptors (RyRs). By combining confocal imaging with simultaneous patch-clamp recording we related [Ca²⁺]_i dynamics to the kinetics of mI_cat and evaluated the effect of RyR- and IP₃R-mediated Ca²⁺ release on mI_cat. In SMCs loaded with the high-affinity Ca²⁺ indicator fluo-3 and voltage-clamped at −50 mV, initiation of CCh (10 μM)-induced Ca²⁺ waves preceded the appearance of mI_cat (n = 7), which reached a peak about 1 s after the maximal increase in [Ca²⁺]_i was observed (n = 17). Neither spontaneous Ca²⁺ sparks (n = 8) nor caffeine (5 mM)-induced (n = 14) RyR-mediated Ca²⁺ release had any effect on mI_cat. The latter cannot be explained by an equal but opposing inhibitory effect of caffeine on muscarinic cationic channels since application of 5 mM caffeine did not inhibit mI_cat when [Ca²⁺]_i was strongly buffered with Ca²⁺/BAPTA buffer (n = 5). Nor can it be attributed to an effect of caffeine on other mechanisms possibly involved in the regulation of Ca²⁺ sensitivity of muscarinic cationic channels since in the presence of 5 mM caffeine, flash-release of Ca²⁺ upon cell dialysis with 5 mM NP-EGTA/3.8 mM Ca²⁺ potentiated mI_cat in the same way as in control (n = 5). In contrast, flash-release of IP₃ (30 μM of ‘caged’ IP₃ in the patch pipette) augmented mI_cat (n = 15). The kinetics of the flash-induced current closely followed the dynamics of intracellular [Ca²⁺]_i changes and a threshold [Ca²⁺]_i for activation of mI_cat in this case was much lower than that observed upon flash-release of Ca²⁺ (n = 10) when responses of fluo-3 to the rise in [Ca²⁺]_i started to develop and saturated prior to that of mI_cat, thus reflecting that muscarinic cationic channels have a lower affinity to Ca²⁺ than fluo-3. Possible activation of muscarinic cationic channels by IP₃ directly or by depletion of IP₃-sensitive Ca²⁺ store was ruled out, as flash-release of IP₃ failed to augment mI_cat when [Ca²⁺]_i was strongly buffered with Ca²⁺/BAPTA buffer (n = 4) or Ca²⁺ store was depleted with 0.1 μM thapsigargin (n = 5). Intracellular calcium store visualised with the low-affinity Ca²⁺ indicator fluo-3FF consisted of the sub-plasmalemmal sarcoplasmic reticulum (SR) and some perinuclear formation (n = 35). Immunostaining revealed that type1 IP₃Rs are predominant in sub-plasmalemmal SR (n = 40) while RyRs are confined to the central region of the cell (n = 18). In non-patched SMCs loaded with fluo-4 AM, CCh evoked staircase-like increase in [Ca²⁺]_i (consistent with summation of APs) with an initial sub-plasmalemmal [Ca²⁺]_i rise in regions where SR was found (n = 19). This pattern of [Ca²⁺]_i dynamics persisted after inhibition of RyRs with 100 μM ryanodine (n = 4), but not after block of VGCCs with 10 μM nicardipine (n = 6) or IP₃Rs with 30 μM 2-APB (n = 3). These results suggest that: (1) IP₃Rs are major contributor to the [Ca²⁺]_i rise upon muscarinic receptor activation, (2) IP₃R-mediated Ca²⁺ release is potentiated by Ca²⁺ entry through VGCCs and plays a central role in the modulation of mI_cat and (3) IP₃ may sensitise muscarinic cationic channels to Ca²⁺.