In gastrointestinal smooth muscles, M₂ and M₃ muscarinic receptors exist with a preponderance of the former subtype. These receptors are believed to mediate the action of the parasympathetic neurotransmitter acethylcholine. However, pharmacological analyses of the contractile responses to muscarinic agonists including carbachol (CCh) have revealed that they are mediated extensively by the minor M₃ subtype; the role of M₂ subtype is unclear. To explore the role of M₂ and M₃ subtypes in the muscarinic contraction, we characterized isometric contractions to CCh in ileal longitudinal smooth muscle strips from humanely killed wild-type mice and mice genetically lacking M₂ or M₃ subtypes. Single applications of CCh (0.1-100 μM) produced concentration-dependent contractions in preparations from M₂-knockout (KO) and M₃-KO mice, mediated via M₃ and M₂ subtypes, respectively, as judged by the sensitivity of contractile responses to blockade by the M₂-preferring antagonist methoctramine (300 nM) or the M₃-preferring antagonist 4-DAMP (30 nM). The M₂-mediated contractions were mimicked in shape by submaximal stimulation with high K⁺ (up to 35 mM), almost abolished by voltage-dependent Ca²⁺ channel (VDCC) antagonists (n=8) or depolarization with 140 mM K⁺ medium (n=7), and greatly reduced by pertussis toxin (PTX) treatment; the maximum contractile response to CCh expressed as a percentage of the 70 mM K⁺ value in PTX-treated and -untreated preparations was 26.9 ± 6.3% (n=8) and 94.8 ± 4.9% (n=12), respectively, and the difference was statistically significant (unpaired t test: P<0.05). The M₃-mediated contractions were only partially inhibited by VDCC antagonists (82.7 ± 3.4% (n=6) of the control response at 10 μM CCh) or 140 mM K⁺ medium (23.5 ± 4.3% (n=6) of the control response at 10 μM CCh). The contractions observed during high K⁺ medium consisted of different components, either sensitive or insensitive to extracellular Ca²⁺. PTX treatment had little or no effect on the M₃-mediated contractions (the maximum contractile response to CCh was 136.2 ± 4.0% (n=6) in PTX-treated and 142.4 ± 9.7% (n=7) in PTX-untreated preparations). The CCh contractions observed with wild type preparations consisted of PTX-sensitive and -insensitive components. The PTX-sensitive component was functionally significant only at low CCh concentrations (0.01 to 3 μM). The results suggest that the M₂ receptor, through PTX-sensitive mechanisms, induces ileal contractions which depend on voltage-dependent Ca²⁺ entry, especially associated with action potential discharge, and that the M₃ receptor, through PTX-insensitive mechanisms, induces contractions which depend on voltage-dependent and -independent Ca²⁺ entry and intracellular Ca²⁺ release. In intact tissues co-expressing M₂ and M₃ receptors, M₂ receptor activity appears functionally relevant only when fractional receptor occupation is relatively small.