The synaptotagmin family has been implicated in calcium-dependent neurotransmitter release, although Synaptotagmin 1 is the only isoform demonstrated to control synaptic vesicle fusion. Here, we report the characterization of the six remaining synaptotagmin isoforms encoded in the Drosophila genome, including homologues of mammalian Synaptotagmins 4, 7, 12, and 14. Like Synaptotagmin 1, Synaptotagmin 4 is ubiquitously present at synapses, but localizes to the postsynaptic compartment. The remaining isoforms were not found at synapses (Synaptotagmin 7), expressed at very low levels (Synaptotagmins 12 and 14), or in subsets of putative neurosecretory cells (Synaptotagmins α and β). Consistent with their distinct localizations, overexpression of Synaptotagmin 4 or 7 cannot functionally substitute for the loss of Synaptotagmin 1 in synaptic transmission. Our results indicate that synaptotagmins are differentially distributed to unique subcellular compartments. In addition, the identification of a postsynaptic synaptotagmin suggests calcium-dependent membrane-trafficking functions on both sides of the synapse.