1. When a nerve-muscle preparation is paralysed by tetrodotoxin, brief depolarizing pulses applied to a motor nerve ending cause packets of acetylcholine to be released and evoke end-plate potentials (e.p.p.s), provided calcium ions are present in the extracellular fluid.2. By ionophoretic discharge from a 1 M-CaCl(2) pipette, it is possible to produce a sudden increase in the local calcium concentration at the myoneural junction, at varying times before or after the depolarizing pulse.3. A brief application of calcium facilitates transmitter release if it occurs immediately before the depolarizing pulse. If the calcium pulse is applied a little later, during the period of the synaptic delay, it is ineffective.4. It is concluded that the utilization of external calcium ions at the neuromuscular junction is restricted to a brief period which barely outlasts the depolarization of the nerve ending, and which precedes the transmitter release itself.5. The suppressing effect of magnesium on transmitter release was studied by a similar method, with ionophoretic discharges from a 1 M-MgCl(2)-filled pipette. The results, though not quite as clear as with calcium, indicate that Mg pulses also are only effective if they precede the depolarizing pulses.