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 (mI_Cat) as small as ~10% of mI_Cat 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 mI_Cat each showed a U-shaped current-voltage (I-V) relationship, whereas the M₂-KO mI_Cat 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 O = 0.55 and 0.23 msec). In contrast, active M₃-KO patches showed only 70-pS channel activity, which had three open states (O = 0.55, 3.1 and 17.4 msec). 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 (O = 0.62, 2.7, 16.9 and 121.1 msec), and patch current of this channel activity showed a U-shaped I-V curve similar to the WT mI_Cat. 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 mI_Cat generation. The delineation of this pathway is consistent with the formation of a functional unit by the M₂/G_o protein and the M₃/PLC systems predicted to control cationic channels.