Parasympathetic control of gastrointestinal smooth muscles (SM) is mediated via muscarinic (M2/M3) receptors, activation of which causes smooth muscle cell (SMC) depolarization via opening of muscarinic cationic channels (MCCs). Plasma membrane (PM) depolarization increases the action potential (AP)frequency thus facilitating Ca²⁺ entry via voltage-gated Ca²⁺ channels (VGCCs). Activation of M2 receptors gates MCCs via G_oα. Activation of M3 receptors stimulates PLC-β, which hydrolyses PIP₂ to DAG and IP₃. MCC opening is synergistically facilitated by intracellular Ca²⁺ and IP₃ [1]. As inhibitors of VGCCs greatly attenuate cholinergic contraction, it was suggested that Ca²⁺ entry via VGCCs is the major mechanism of Ca²⁺ mobilisation. Analysis of the mechanisms of the carbachol (CCh) – induced Ca²⁺ mobilisation brought us to revision of this idea. The experiments were conducted on SMCs freshly isolated from the longitudinal layer of the guinea-pig ileum. Using fast confocal imaging we analysed [Ca²⁺]_i changes following stimulation of M2/M3 receptors with CCh and related the spatial pattern of CCh-induced [Ca²⁺]_i transients to the intracellular distribution of IP₃ receptors (IP₃Rs) and ryanodine receptors (RyRs). By combining Ca²⁺ imaging with tight-seal recording we related the changes in [Ca²⁺]_i to the changes in the cell membrane potential. Using pharmacological tools we analysed the contribution of VGCCs, IP₃Rs and RyRs to CCh-induced [Ca²⁺]_i transients. The effect of some of these agents on VGCCs, MCCs, CCh-induced depolarization and SM contraction was tested. CCh-induced APs were associated with abrupt increases of [Ca²⁺]_i (Ca²⁺ spikes) at sub-PM SR elemets enriched with IP3Rs and poor in RyRs. Summation of Ca²⁺ spikes gave rise to an initial [Ca²⁺]_i transient followed by a delayed phase/[Ca²⁺]_i oscillations. Block of RyRs (50-100 μM tetracaine or 100 μM ryanodine) abolished the delayed phase but had little effect on Ca²⁺ spikes/initial [Ca²⁺]_i transient. Block of VGCCs (5 μM nicardipine) or IP₃Rs (2 μM xestospongin C or 30 μM 2-APB) suppressed both the initial and delayed phases of the [Ca²⁺]_i transient and abolished Ca²⁺ spikes. Current through VGCCs was partially inhibited by xestospongin C, but was slightly augmented by 2-APB. 2-APB partially inhibited MCCs but not CCh-induced depolarization. Cholinergic contraction was strongly attenuated by 2-APB or tetracaine. Thus, genesis of Ca²⁺ spikes by VGCCs involves IP₃Rs-mediated Ca²⁺-release. Both IP₃Rs and RyRs are engaged in the full-scale contractile response to CCh.