Acetylcholine regulates bronchial tone in airway smooth muscle (ASM) by binding to both M2 and M3 muscarinic receptors (Fisher et al., 2004), however, the mechanisms underlying M2R-mediated contractions of ASM are still unclear. Recent studies have indicated that Kv7 channels are functionally expressed in ASM and that Kv7 currents are suppressed by cholinergic agonists (Brueggemann et al., 2012). The purpose of the present study was to investigate if M2R-mediated contractions of ASM were affected by Kv7 channel modulators. Adult male and female C57BL/6 mice were euthanized by intraperitoneal injection of pentobarbital (100 mg/kg) and the lungs were removed to obtain primary bronchial rings. The bronchial rings were mounted into organ baths and isometric tension recordings were employed to examine the effect of M2R antagonists and Kv7 channel modulators on contractions induced by application of the cholinergic agonist carbachol (CCh). The M2 muscarinic antagonist methoctramine (100 nM) reduced the mean contraction amplitude of responses induced by CCh (100 nM) by 68% from 5751 ± 768 to 1227 ± 624 mN.s (n=6, p<0.0001, two way ANOVA, Bonferroni’s multiple comparisons test). The EC50 for CCh was shifted from 100 to 278 nM in methoctramine (n=6, CI 95%: 75 – 160 nM and 234 – 333 nM, respectively). Similarly, the Kv7 channel activator retigabine (10 µM) reduced the amplitude of CCh-induced contractions, and increased the EC50 from 90 to 240 nM (n=6, CI 95%: 60 – 127 nM and 190-304 nM, respectively). The M3R antagonist 4-DAMP (3 nM) reduced the amplitude of CCh-induced contractions (300 nM) by 69% and the residual contractions were abolished by methoctramine (n=9, p<0.001, two tailed Student’s t-test). 4-DAMP-resistant CCh responses were also abolished by retigabine (n=10, p<0.01, two tailed Student’s t-test) and enhanced by the Kv7 channel blocker XE-991 (n=28, p<0.0001, ANOVA, Bonferroni’s multiple comparisons test). These data suggest that; 1) M2Rs may have a greater functional role in ACh-induced contractions of ASM than previously realised and 2) that M2R-dependent responses may involve Kv7 channels.