Studies have shown that the activity of muscarinic receptors and their affinity to agonists are sensitive to membrane potential. It was reported that in airway smooth muscle (ASM) depolarization evoked by high K+ solution increases contractility through direct effects on M3 muscarinic receptors. In this study, we assessed the physiological relevance of voltage sensitivity of muscarinic receptors on ASM contractility. Our findings reveal that depolarization by high K+ solution induces contraction in intact mouse trachea predominantly through activation of acetylcholine release from embedded nerves, and to a lesser extent by direct effects on M3 receptors. We therefore devised a pharmacological approach to depolarize tissue to various extents in an organ bath preparation, and isolate contraction due exclusively to ASM muscarinic receptors within range of physiological voltages. Our results indicate that unliganded muscarinic receptors do not contribute to contraction regardless of voltage. Utilizing low K+ solution to hyperpolarize membrane potentials during contractions had no effect on liganded muscarinic receptor-evoked contractions, although it eliminated the contribution of voltage-gated calcium channels. However, we found that muscarinic signaling was potentiated by at least 42% at depolarizing voltages (average -12 mV) induced by high K+ solution (20 mmol/L K+ ). In summary, we conclude that contractions evoked by direct activation of muscarinic receptors have negligible sensitivity to physiological voltages. However, contraction activated by cholinergic stimulation can be potentiated by membrane potentials occurring beyond the physiological range of ASM.