Membrane depolarization is the signal that triggers release of neurotransmitter from nerve terminals. As a result of depolarization, voltage-dependent Ca2+ channels open, level of intracellular Ca2+ increases, and release of neurotransmitter commences. Previous study had shown that in rat brain synaptosomes, muscarinic acetylcholine (ACh) receptors (mAChRs) interact with soluble NSF attachment protein receptor proteins of the exocytic machinery in a voltage-dependent manner. It was suggested that this interaction might control the rapid, synchronous release of acetylcholine. The present study investigates the mechanism for such a voltage-dependent interaction. Here we show that depolarization shifts mAChRs, specifically the m2 receptor subtype, to a low affinity state toward its agonists. At resting potential, mAChRs are in a high affinity state (Kd of ~20 nM) and they shift to a low affinity state (K(d) of tens of μM) upon membrane depolarization. In addition, interaction between m2 receptor subtype and the exocytic machinery increases with receptor occupancy. Both phenomena are independent of Ca2+ influx. We propose that these results may explain control of ACh release from nerve terminals. At resting potential the exocytic machinery is clamped due to its interaction with the occupied mAChR and depolarization relieves this interaction. This, together with Ca2+ influx, enables release of ACh to commence.