The role of presynaptic muscarinic autoreceptors in the initiation of phasic acetylcholine release at frog and mouse neuromuscular junctions was studied by measuring the dependence of the amount (m) of acetylcholine release on the level of presynaptic depolarization. Addition of methoctramine, (a blocker of M₂ muscarinic receptors), or of acetylcholinesterase, increased release in a voltage-dependent manner; enhancement of release declined as depolarizing pulse amplitude increased. In frogs and wild-type mice the slope of log m/log pulse amplitude (PA) was reduced from ~7 in the control to ~4 in the presence of methoctramine or acetylcholinesterase. In M₂ muscarinic receptor knockout mice, the slope of log m/log PA was much smaller (~4), and was not further reduced by addition of either methoctramine or acetylcholinesterase. The effect of a brief (0.1 ms), but strong (-1.2 µA), depolarizing prepulse on the dependence of m on PA was also studied. The depolarizing prepulse had effects similar to those of methoctramine and acetylcholinesterase. In particular, it enhanced release of test pulses in a voltage-dependent manner, and reduced the slope of log m/log PA from ~7 to ~4. Methoctramine + acetylcholinesterase occluded the prepulse effects. In knockout mice, the depolarizing prepulse had no effects. The cumulative results suggest that initiation of phasic acetylcholine release is achieved by depolarization-mediated relief of a tonic block imposed by presynaptic M₂ muscarinic receptors.