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tor proteins. The C2B domain mediates Ca2+-triggered synaptotagmin oligomerization, binds AP-2 and is important for the interaction of synaptotagmin with Ca2+ channels. Here, we report that these are conserved biochemical properties: Ca2+ promoted the hetero-oligomerization of synaptotagmin I with synaptotagmins III and IV, and all three synaptotagmin isoforms bound the synprint region of the alpha1B subunit of N-type Ca2+ channels. Using chimeric and truncated C2 domains, we defined a common region of C2B that mediates oligomerization and AP-2 binding. Within this region, two adjacent lysine residues were identified that were critical for synaptotagmin oligomerization, AP-2, and synprint binding. Competition experiments demonstrated that the synprint fragment was an effective inhibitor of synaptotagmin oligomerization and also blocked binding of synaptotagmin to AP-2. In a model for the structure of C2B, the common effector binding site localized to a putative Ca2+-binding loop and a concave region formed by two beta-strands. These studies provide the first structural information regarding C2B target protein recognition and provide the means to selectively disrupt synaptotagmin-effector interactions for functional studies.