The influences of synaptic plasma membranes on release of acetylcholine (ACh) from synaptic vesicles isolated from rat brain were examined. In the presence of ATP, Mg++ and Ca++ but absence of cytoplasm from the nerve endings, the synaptic plasma membranes did not increase ACh release indicating absence of a stimulating factor which is known to be present in the cytoplasm. In presence of ATP, Mg++, Ca++ and the cytoplasm, the synaptic plasma membranes inhibited ACh release from the synaptic vesicles in high K+ medium, though not in high Na+ medium. Binding of Ca++ by the synaptic plasma membranes was dependent on ATP, inhibited by Na+ and stimulated by K+. Thus, the synaptic plasma membranes may inhibit ACh release in high K+ medium due to reduction in the concentration of free Ca++.