The GTPase dynamin I is required for synaptic vesicle (SV) endocytosis. Our observation that dynamin binds to the SV protein synaptophysin in a Ca(2+)-dependent fashion suggested the possibility that a dynamin/synaptophysin complex functions in SV recycling. In this paper we show that disruption of the dynamin/synaptophysin interaction by peptide injection into the squid giant synapse preterminal results in a decrease in transmitter release during high-frequency stimulation, indicating an inhibition of SV recycling. Electron microscopy of these synapses reveals a depletion of SVs, demonstrating a block of vesicle retrieval after fusion. In addition, we observed an increase in clathrin-coated vesicles, indicating that the peptide does not block clathrin-dependent endocytosis. We conclude that the dynamin/synaptophysin complex functions in a clathrin-independent mechanism of SV endocytosis that is required for efficient synaptic transmission.