Reliable neuronal communication depends on accurate temporal correlation between the action potential and neurotransmitter release. Although a requirement for Ca2+ in neurotransmitter release is amply documented, recent studies have shown that voltage-sensitive G protein-coupled receptors (GPCRs) are also involved in this process. However, how slow-acting GPCRs control fast neurotransmitter release is an unsolved question. Here we examine whether the recently discovered fast depolarization-induced charge movement in the M2-muscarinic receptor (M2R) is responsible for M 2R-mediated control of acetylcholine release. We show that inhibition of the M2R charge movement in Xenopus oocytes correlated well with inhibition of acetylcholine release at the mouse neuromuscular junction. Our results suggest that, in addition to Ca2+ influx, charge movement in GPCRs is also necessary for release control.