Cortical granules are stimulus-dependent secretory vesicles found in the egg cortex of most vertebrates and many invertebrates. Upon fertilization, an increase in intracellular calcium levels triggers cortical granules to exocytose enzymes and structural proteins that permanently modify the extracellular surface of the egg to prevent polyspermy. Synaptotagmin is postulated to be a calcium sensor important for stimulus-dependent secretion and to test this hypothesis for cortical granule exocytosis, we identified the ortholog in two sea urchin species that is present selectively on cortical granules. Characterization by RT-PCR, in-situ RNA hybridization, Western blot and immunolocalization shows that synaptotagmin I is expressed in a manner consistent with it having a role during cortical granule secretion. We specifically tested synaptotagmin function during cortical granule exocytosis using a microinjected antibody raised against the entire cytoplasmic domain of sea urchin synaptotagmin I. The results show that synaptotagmin I is essential for normal cortical granule dynamics at fertilization in the sea urchin egg. Identification of this same protein in other developmental stages also shown here will be important for interpreting stimulus-dependent secretory events for signaling throughout embryogenesis. © 2006 Wiley-Liss, Inc.