N-type Ca2+ channels bind directly to the synaptic core complex of VAMP/synaptobrevin, syntaxin, and SNAP-25. Peptides containing the synaptic protein interaction ("synprint") site caused dissociation of N-type Ca2+ channels from the synaptic core complex. Introduction of synprint peptides into presynaptic superior cervical ganglion neurons reversibly inhibited synaptic transmission. Fast EPSPs due to synchronous transmitter release were inhibited, while late EPSPs arising from asynchronous release following a train of action potentials were increased and paired-pulse facilitation was increased. The corresponding peptides from L-type Ca2+ channels had no effect, and the N-type peptides had no effect on Ca2+ currents through N-type Ca2+ channels. These results are consistent with the hypothesis that binding of the synaptic core complex to presynaptic N-type Ca2+ channels is required for Ca2+ influx to elicit rapid, synchronous neurotransmitter release.