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Using mutant mice genetically lacking certain subtypes of muscarinic receptor, we have studied muscarinic signal pathways mediating cationic channel activation in intestinal smooth muscle cells. In cells from M2 subtype-knockout (M2-KO) or M3-KO mice, carbachol (100 μm) evoked a muscarinic cationic current (mICat) as small as ∼10% of mICat in wild-type (WT) cells. No appreciable current was evoked in M2/M3 double-KO cells. All mutant type cells preserved normal G-protein–cationic channel coupling. The M3-KO and WT mICat each showed a U-shaped current–voltage (I–V) relationship, whereas the M2-KO mICat displayed a linear I–V relationship. Channel analysis in outside-out patches recognized 70-pS and 120-pS channels as the major muscarinic cationic channels. Active patches of M2-KO cells exhibited both 70-pS and 120-pS channel activity usually together, either of which consisted of brief openings (the respective mean open times Oτ= 0.55 and 0.23 ms). In contrast, active M3-KO patches showed only 70-pS channel activity, which had three open states (Oτ= 0.55, 3.1 and 17.4 ms). In WT patches, besides the M2-KO and M3-KO types, another type of channel activity was also observed that consisted of 70-pS channel openings with four open states (Oτ= 0.62, 2.7, 16.9 and 121.1 ms), and patch current of this channel activity showed a U-shaped I–V curve similar to the WT mICat. The present results demonstrate that intestinal myocytes are endowed with three distinct muscarinic pathways mediating cationic channel activation and that the M2/M3 pathway targeting 70-pS channels, serves as the major contributor to mICat generation. The delineation of this pathway is consistent with the formation of a functional unit by the M2-Go protein and the M3-PLC systems predicted to control cationic channels.