Activation of muscarinic receptors, the major excitatory receptors involved in the parasympathetic control of smooth muscle function, produces membrane depolarization through the cationic channel opening. Ca²⁺ mobilized from intracellular stores in response to the activation of M₃ receptors potentiates muscarinic cationic current (mI_cat). We therefore related the dynamics of carbachol (CCh)-induced [Ca²⁺]_i changes to the kinetics of mI_cat and evaluated the effect of Ca²⁺ release through ryanodine receptors (RyRs) and IP₃ receptors (IP₃Rs) on mI_cat by combining confocal imaging of [Ca²⁺]_i with simultaneous patch-clamp recording of mI_cat.The experiments were carried out on myocytes freshly isolated from the guinea-pig ileum. The animals were killed by decapitation after cervical dislocation as approved under Schedule 1 of the UK Animals (Scientific Procedures) Act 1986. Fast x-y confocal imaging of the myocytes loaded with Ca²⁺-sensitive indicator fluo-3 revealed that CCh (10 µM)-induced Ca²⁺ waves propagated from the cell ends towards the myocyte centre at 45.9±8.8 µm s^-1 (mean±S.E.M., n=13) and that Ca²⁺ wave initiation preceded any measurable mI_cat by 229±55 ms (n=7). CCh-induced [Ca²⁺]_i transient peaked 1.22±0.11 s (n=17) before mIreached its maximum. At −50 mV, spontaneous release of Ca²⁺ through RyRs resulting in Ca²⁺ sparks had no effect on CChinduced mI_cat but activated BK channels leading to spontaneous transient outward currents. Ca²⁺ release through RyRs induced by brief application of 5 mM caffeine was initiated at the cell centre but did not augment mI_cat (n=14). The latter was due neither to an inhibition of the cationic channels by caffeine (since application of 5 mM caffeine did not inhibit mI_cat,when [Ca²⁺]_i was clamped with Ca²⁺/BAPTA buffer) nor to an effect of caffeine on other mechanisms of the cationic channel Ca²⁺-sensitivity (since in the presence of 5 mM caffeine, intracellular photorelease of Ca²⁺ potentiated mI_cat in the same way as in control). Intracellular photorelease of IP₃ augmented mI_cat (n=15) at lower [Ca²⁺]_i than required for potentiation of mI_cat by Ca²⁺ alone (n=10). Intracellular calcium store visualised with a low-affinity Ca²⁺ indicator fluo-3FF (n=35) consisted of the superficial sarcoplasmic reticulum (SR) network and some perinuclear formation interconnected with the superficial SR. Immunostaining of the myocytes with antibodies to IP₃Rs (n=40) and to RyRs (n=18) revealed that type1 IP₃Rs are predominant in the superficial SR while RyRs are confined to the central region of the cell. These results suggest that IP₃R-mediated Ca²⁺ release plays a central role in the modulation of mI_cat in the guinea-pig ileum and that IP₃ may sensitise to Ca²⁺ the regulatory mechanisms of the muscarinic cationic channels opening.