Postjunctional M2 muscarinic receptors (M2Rs) on airway smooth muscle (ASM) outnumber M3 receptors (M3Rs) by a ratio of 4:1 in most species, yet it is the M3Rs that are thought to mediate the bronchoconstrictor effects of acetylcholine. However, it is now known that stimulation of M2Rs causes a profound enhancement of M3R-mediated ASM contractions induced by low frequency stimulation (2 Hz) and when the stimulus interval was decreased from 100 to 10 s [1]. The purpose of the present study was to investigate the cellular mechanisms responsible for M2R-dependent potentiation of nerve-evoked contractions of murine ASM. Contractions of murine bronchial rings were induced by electric field stimulation (EFS) and recorded using isometric tension recording. Reduction in stimulus interval from 100 to 10 s increased mean contraction amplitude from 0.8 ± 0.1 to 1.6 ± 0.2mN (N=6, p<0.0001). Subsequent addition of the L-type Ca²⁺ channel (LTCC) blocker, nifedipine (1 µM) reduced mean contraction amplitude to 0.83 ± 0.14mN (N=6, p<0.0001). In contrast, contractions evoked at 100 s intervals, that are exclusively mediated by M3Rs, were unaffected by nifedipine. Previous studies on canine ASM found that Ca²⁺ influx via LTCC was buffered by the activity of superficial sarcoplasmic/endoplasmic reticulum ATPase (SERCA) [2]. We hypothesised that M2R-dependent contractions of ASM resulted from reduced buffering of Ca²⁺ entry via LTCC by SERCA. Quantitative polymerase chain reaction (qPCR) analyses revealed that SERCA2b exhibited the highest expression among SERCA isoforms in murine bronchi tissues and staining of isolated ASM cells with SERCA2 antibodies using immunocytochemistry showed that SERCA2 was located at the periphery of ASM cells, adjacent to the plasma membrane. Inhibition of SERCA with thapsigargin (1 µM) increased the mean amplitude of nerve-evoked contractions of ASM, at 100 s intervals, from 1.4 ± 0.1 to 3.2 ± 0.3 mN (N=6, p<0.001) and these effects were also reversed by nifedipine which lowered the amplitude to 0.7 ± 0.1mN (N=6, p<0.001). Thapsigargin also increased the area of ASM contractions induced by the LTCC activator FPL64176 (1 µM), from 776 ± 221 to 2686 ± 643 mN.s (N=6, p<0.05). Similar to the effects of nifedipine, the Ano1 channel blocker Ani9 (1 µM) abolished M2R-dependent enhancement of EFS-evoked contractions at 10 s intervals (from 1.4 ± 0.1 to 0.44 ± 0.1 mN (N=6, p<0.01), but did not affect responses at 100 s intervals. Ani9 also reversed the stimulatory effects of thapsigargin on EFS-evoked contractions at 100 s. Thapsigargin increased mean contraction amplitude from 0.9 ± 0.1 to 1.9 ± 0.1mN (N=6, p<0.01) and Ani9 reduced contraction amplitude to 0.7 ± 0.1 mN (N=6, p<0.001). These data indicate that M2R-mediated potentiation of cholinergic-nerve mediated contractions of ASM relies on activation of Ano1 channels and Ca²⁺ influx via LTCC by a mechanism involving inhibition of buffering of Ca²⁺ influx by SERCA.