Muscarinic receptor cation current in gastrointestinal smooth muscles is strongly modulated by both external and intracellular Ca²⁺, but the mechanisms of this dependence at the single channel level have not been studied yet. In single ileal single smooth muscle cells, isolated from humanely killed mice, external application of 50 μM carbachol (CCh) or internal application of 200 μM GTPγS evoked an inward current of 0.60±0.05 nA at −40 mV (n=50). The current reversed close to 0 mV and its current-voltage relationship was U-shaped at negative potentials. Single channel recordings in outside-out patches revealed that CCh-induced current was carried via two types of monovalent selective cation channels with unitary conductances of about 17 and 70 pS (n=12). The behaviour of the 70-pS channel was consistent with the whole-cell muscarinic current properties implying its major contribution to the integral current. We therefore investigated the effects of changes in the external (2.5 or 10 mM) and internal (30, 100, 500 nM) Ca²⁺ concentration on the single 70 pS-channel properties. Ca²⁺ added to a nominally Ca²⁺-free external solution at 2.5 or 10 mM reduced the unitary channel conductance from 70 pS to 46 pS and 42 pS, respectively, but had no significant effect on its open probability (P_O). When [Ca²⁺]_i was clamped at 30, 100, or 500 nM using a BAPTA-Ca²⁺ mixture the amplitude of the CCh- or GTPγS-evoked whole-cell current at 100 nM [Ca²⁺]_i was several times greater compared with either 30 or 500 nM [Ca²⁺]_i. Correspondingly, with 30 or 500 nM Ca²⁺ in the pipette solution P_O of the 70 pS-channel was reduced from 0.2-0.5 (at 100 nM) to 0.02-0.1. In contrast, the variations in [Ca²⁺]_i had no effect on the unitary conductance. Using the fura-2-based calcium imaging system and simultaneous patch-clamp current recordings we found that the most potent blocker of the 70-pS single channel activity, quinine (20 μM), produced a strong decrease of the steady-state component of the CCh-evoked [Ca²⁺]_i rise. Other common blockers of various cation channels, SK&F 96365, LOE908, 2-APB and La³⁺ applied at micromolar concentrations, produced similar effects. It is concluded that in murine ileal smooth muscle cells the 70-pS cation channel mainly mediates cholinergic depolarisation and is important for maintaining the elevated intracellular calcium concentration during the activation of the muscarinic receptors.