The method of external focal recording from the neuromuscular junction has been used to locate the site of action of calcium ions in the transmission process. The muscle is placed in a calcium-deficient medium (which contains magnesium as a substitute), and the effect of localized calcium application from the recording micropipette is studied. Electrophoretic application of calcium is followed within less than 1 s by increased terminal release of acetylcholine, shown by a large increase in the number of quantal components of the end-plate potential. This effect is observed even under conditions when the terminal axon spike diminishes in size during the application of calcium. It is concluded that the action of calcium is concerned directly with the release of the transmitter, and not indirectly-as is sometimes suggested-by facilitating propagation or increasing the amplitude of the terminal nerve spike.