Muscarinic receptors play key roles in the control of gastrointestinal smooth muscle activity. However, specific physiological functions of each subtype remain to be determined. Single cell RT-PCR experiments showed that all five subtypes of muscarinic receptors were present in circular smooth muscle cells of the guinea-pig gastric antrum. Nonselective cation channels (NSCC) activated by ACh or CCh are coupled to pertussis toxin (PTX)-sensitive Go protein through m4 subtype as well as m2 and m3 subtypes in guinea-pig stomach. CCh-activated currents (I_CCh), especially the steady-state I-V relationship of I_CCh showed a chracteristic U-shaped curve; reversal potential of around 0 mV and inward rectification at around +15 mV and a negative slope conductance at negative potential range. Under physiological conditions, the measured single channel conductance of NSCC was approximately 25 pS. The single channel conductance was modulated by external monovalent and divalent cations including Na⁺, Cs⁺, Li⁺, and Ca²⁺ through changing both the open probability and unitary conductance. Through the NSCC, Ca²⁺ can move into the cell from extracellular solution as well as Na⁺. Calculated fractional Ca²⁺ current of I_CCh (f_Ca) was around 1% at the 2 mM [Ca²⁺]_o and at the 4 mM [Ca²⁺]_o, f_Ca was 2.3%. Quinidine blocked I_CCh potently in a reversible manner; IC₅₀ was 0.25 μM. There were two kinds of I_CCh modulations through Ca²⁺-dependent pathways in guinea-pig gastric smooth muscle cells; 1) Facilitation of I_CCh via Ca²⁺/CaM-dependent MLCK pathway, 2) Desensitization of I_CCh via Ca²⁺-dependent PKC pathway. In the mouse stomach, all seven types of TRPC mRNA were detected with RT-PCR. On the basis of electrophysiological, pharmacological, and molecular biological experiments, we reported the mTRPC5 as a candidate for the NSCC activated by muscarinic stimulation in mouse stomach.