Smooth muscle cells of guinea pig ileum express both M2 and M3 subtypes of muscarinic receptors. Under voltage clamp, activation of the muscarinic receptors with carbachol (CCh) induces Ca²⁺-activated K⁺ current (I_K-Ca) and nonselective cationic current (I_cat). Receptor subtypes mediating the current responses were characterized by using pirenzepine, AF-DX116, 4-DAMP and atropine, which have different profiles of the affinity constants for muscarinic receptor subtypes. The muscarinic antagonists inhibited either CCh-evoked I_K-Ca or I_cat with different potencies. Their relative potencies for I_K-Ca and I_cat inhibition resembled the relative affinity constants for M3 and M2 subtypes, respectively. Thus, the I_K-Ca is mediated via the M3 subtype and the I_cat via the M2 subtype.