1. An electrophysiological analysis has been made of the storage of acetylcholine (ACh) in the preganglionic nerve terminals of the isolated superior cervical ganglion of the guinea-pig. The mean amplitude of excitatory post-synaptic potentials recorded intracellularly was taken as a measure of the ACh output per impulse from the terminals of a preganglionic axon.2. Prolonged repetitive stimulation of the cervical sympathetic trunk at 10 and 20 Hz in the presence of hemicholinium No. 3 led to an exponential decline of ACh output as the transmitter formed before the beginning of stimulation was depleted.3. The rate of decline of ACh output during stimulation at 20 Hz (tau = 0.83-1.95 min) was about twice as fast as that at 10 Hz (tau = 1.00-6.83 min).4. The results suggest that, during prolonged stimulation, ACh is released from a single store in the preganglionic nerve terminal and that each impulse releases a constant fraction of the store of the transmitter.