1. Macropatch recording was used to study release of acetylcholine in the frog neuromuscular junction evoked by either direct local depolarization or by an action potential. 2. The quantal content was established by directly counting the released quanta. The time course of release was obtained by constructing synaptic delay histograms. 3. Perfusion of the neuromuscular junction with methoctramine, a selective M 2/M 4 muscarinic antagonist, increased the quantal content and slowed the exponential decay of the synaptic delay histograms. Addition of the agonist muscarine reversed these effects. 4. Addition of acetylcholinesterase prolonged the decay of the delay histogram, and muscarine reversed this effect. 5. Methoctramine slowed the rise time of the postsynaptic current produced by axon stimulation without affecting either the excitatory nerve terminal current or the presynaptic Ca 2+ current. 6. These results show that presynaptic M 2 muscarinic receptors are involved in the process which terminates evoked ACh release.