Presynaptic injection of inositol 1,3,4,5-tetraphosphate, inositol 1,3,4,5,6-pentakisphosphate, or inositol 1,2,3,4,5,6-hexakisphosphate--which we denote here the inositol high-polyphosphate series (IHPS)--is shown to block synaptic transmission when injected into the preterminal of the squid giant synapse. This effect is not produced by injection of inositol 1,4,5-trisphosphate. The synaptic block is characterized by a time course in the order of 15-45 min, depending on the injection site in the preterminal fiber; the fastest block occurs when the injection is made at the terminal release site. Presynaptic voltage clamp during transmitter release demonstrates that IHPS block did not modify the presynaptic inward, calcium current. Analysis of synaptic noise at the postsynaptic axon shows that both the evoked and spontaneous transmitter release are blocked by the IHPS. Tetanic stimulation of the presynaptic fiber at frequencies of 100 Hz indicates that block is accompanied by gradual reduction of the postsynaptic response, demonstrating that the block interferes with vesicular fusion rather than with vesicular docking. These results, in combination with the recently demonstrated observation that the IHPS bind the C2B domain in synaptotagmin [Fukada, M., Aruga, J., Niinobe, M., Aimoto, S. & Mikoshiba, K. (1994) J. Biol. Chem. 269, 29206-29211], suggest that IHPS elements are involved in vesicle fusion and exocytosis. In addition, a scheme is proposed in which synaptotagmin triggers transmitter release directly by promoting the fusion of synaptic vesicles with the presynaptic plasmalemma, in agreement with the very rapid nature of transmitter release in chemical synapses.