1. The sensitivity of the subsynaptic membrane of twitch muscles of the frog and snake to iontophoretically applied acetylcholine (ACh) was determined. Optimal placement of ACh micropipettes on to the postsynaptic membrane resulted in potentials that were similar, though not identical, to the miniature excitatory post-synaptic potentials (min e.p.s.p.s). A sensitive bio-assay was developed to measure the output of ACh from micropipettes; this allowed an estimate to be made of the upper limit of the number of ACh molecules in a quantum of transmitter that is released from the nerve to produce a min e.p.s.p. 2. The assay to calibrate the output of ACh from micropipettes used the end-plate of the snake muscle as an ACh concentration detector. The end-plate was situated within a few mum of an oil-water interface, and a 0-6 nl. droplet of Ringer solution containing a known concentration of ACh (1 muM or less) was formed in the oil phase. The droplet was brought to the interface and, upon touching it, discharged its contents into the Ringer phase immediately above the end-plate. This resulted in a membrane depolarization that was recorded with an intracellular microelectrode. By applying droplets containing various known ACh concentrations a standard curve was constructed. To measure the ACh output of micropipettes a 0-6 nl. droplet of Ringer solution was suspended in the oil. The ACh pipette tip was inserted into the droplet and several thousand pulses of ACh were then delivered. The ACh content of the test droplet was measured by comparing its effectiveness in depolarizing the end-plate with the standard curve. In this manner the number of ACh molecules released in a single pulse was determined as a function of charge passed through the pipette. The output of ACh was linear and an average of 30,000 molecules of ACh were released per pC. 3. The sensitivity of the subsynaptic membrane to iontophoretically applied ACh, using the linear slopes of dose-response curves, in preparations from frog and snake treated with anticholinesterases was usually about 5 mV/pC. It follows that 6000 molecules of ACh are sufficient to produce a depolarization of 1 mV in the subsynaptic membrane. 4. The mean min e.p.s.p.s of muscle fibres treated with anticholinesterase range from 1 to 3 mV. Since the ACh released from an iontophoretic pipette is less effective than the same amount released from the nerve, it is concluded that a quantum of transmitter consists of less than 10,000 molecules of ACh. 5. It is calculated that for each molecule of ACh released in a quantum there results a minimum net flow of 3000 univalent ions across the synaptic membrane.