Synaptotagmins constitute a large protein family, characterized by one transmembrane region and two C2 domains, and can be classified into several subclasses based on phylogenetic relationships and biochemical activities (Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421-31427). Synaptotagmin I (Syt I), a possible Ca2+ sensor for neurotransmitter release, showed both Ca2+-dependent (via the C2 domain) and -independent (via the NH2-terminal domain) self-oligomerization, which are thought to be important for synaptic vesicle exocytosis. However, little is known about the relationship between these two interactions and the Ca2+-dependent oligomerization properties of other synaptotagmin isoforms. In this study, we first examined the Ca2+-dependent self-oligomerization properties of synaptotagmin family by co-expression of T7- and FLAG-tagged Syts (full-length or cytoplasmic domain) in COS-7 cells. We found that Syt VII is a unique class of synaptotagmins that only showed robust Ca2+-dependent self-oligomerization at the cytoplasmic domain with EC50 values of about 150 μM Ca2+. In addition, Syt VII preferentially interacted with the previously described subclass of Syts (V, VI, and X) in a Ca2+-dependent manner. Co-expression of full-length and cytoplasmic portion of Syts VII (or II) indicate that Syt VII cytoplasmic domain oligomerizes in a Ca2+-dependent manner without being tethered at the NH2-terminal domain, whereas Ca2+-dependent self-oligomerization at the cytoplasmic domain of other isoforms (e.g. Syt II) occurs only when the two molecules are tethered at the NH2-terminal domain.