1. A study has been made of the release of acetylcholine (ACh) from the preganglionic nerve terminals of the isolated superior cervical ganglion of the guinea-pig during short trains of impulses, using the amplitude of the excitatory post-synaptic potential (e.p.s.p.) as a measure of the ACh released by each impulse. 2. The time course of decay of facilitation following a single impulse could be described by two exponential components, with T1 = 200 msec, and T2 = 13-3 sec. The increase in ACh output at the beginning of stimulation at frequencies smaller than or equal to 2Hz was reasonably predicted in terms of summation of the individual facilitatory effects of each impulse in the train, but fell short of the prediction at higher frequencies. 3. The steady-state output of ACh during repetitive stimulation at frequencies between 0-5 and 20 Hz was lower than that predicted by summation of the facilitatory effects of each impulse, but reached the predicted level at frequencies smaller than or equal to 2Hz in raised Mg2+ concentrations. 4. Statistical analysis of the quantal content (m) of e.p.s.p.s evoked by each of the first five impulses in a train showed that Poisson statistics described release of ACh at the beginning of a train in most cases; when binomial statistics could be applied (two of seven axons studied), the increase in m was accompanied by an increase in the statistical parameter, n. 5. Analyses were also made of release during continuous stimulation; at the time when the steady state of release was reached, the statistical parameter, p, had also increased. Increased release of ACh at increased frequencies of stimulation was associated with increases in p in axons with p smaller than 0-5; however, in most axons (eleven of seventeen), p was greater than 0-5 in the steady state, and increases in m with frequency were due to increases in n.