To determine the functional role of presynaptic proteins in the neurotransmitter release, I have employed cholinergic synapses formed between superior cervical ganglion neurons in culture. These synapses expressed proteins characteristic of mature synapses: immunofluorescence staining showed the presence of synaptophysin, synaptotagmin, VAMP/synaptobrevin-2, syntaxin and neurexin. The function of these proteins seems to be similar to that of mature synapses because botulinum neurotoxins A, E and C1 inhibited neurotransmitter release evoked by presynaptic action potentials. With this preparation, I have obtained evidence supporting roles for myosin II and myosin light chain kinase in neurotransmitter secretion. Acetylcholine release was inhibited by introduction of antibody against myosin II or inhibitors of myosin light chain kinase. This evidence suggests a model in which myosin light chain kinase phosphorylates myosin, and the resultant change in actin-myosin interactions is involved in some steps of transmitter release.