Synaptotagmin I, a transmembrane protein located on neuronal vesicles, acts as the Ca2+ sensor for exocytosis. More than sixteen isoforms of synaptotagmin have been identified all of which contain two Ca2+ binding domains, C2A and C2B. The functions of these additional isoforms are not well understood. Limited proteolysis was used to determine if calcium drives conformational changes in isoforms II and III and to calculate the EC50 of calcium binding for these isoforms. It was found that calcium does drive conformational changes and EC50 = 170 uM for synaptotagmin II and EC50 = 45 uM for synaptotagmin III. Our calculated EC50 value for synaptotagmin I was 56 uM which is in agreement with previously published results.