Facilitation of quantal release has been thought to be maximal immediately after the first action potential in the presynaptic terminal. However, at the frog neuromuscular junction at 0 degrees C no facilitation was observed in response to direct nerve depolarization when the interval between stimulus pairs was less than 10 msec (Dudel, 1986), while at intervals of 20 msec and beyond, facilitation was increased (Molgo and Van der Kloot, 1991). In the present experiments, facilitation to pairs of nerve action potentials was measured both by the method of failures and by comparing the total inward postsynaptic current generated by the first and second action potentials. Facilitation was observed at intervals as short as 7 msec, but 20–30 msec was required for facilitation to reach a maximum. This suggests that facilitation requires a second messenger or the action of Ca2+ at a site other than that eliciting exocytosis.