Neuronal exocytosis is mediated by Ca21-triggered rearrangementsbetween proteins and lipids that result in the opening anddilation of fusion pores. Synaptotagmin I (syt I) is a Ca21-sensingprotein proposed to regulate fusion pore dynamics via Ca21-promoted binding of its cytoplasmic domain (C2A-C2B) to effectormolecules, including anionic phospholipids and other copies of syt.Functional studies indicate that Ca21-triggered oligomerization ofsyt is a critical step in excitation–secretion coupling; however, thisactivity has recently been called into question. Here, we show thatCa21 does not drive the oligomerization of C2A-C2B in solution.However, analysis of Ca21zC2A-C2B bound to lipid monolayers,using electron microscopy, revealed the formation of ring-likeheptameric oligomers that are '11 nm long and '11 nm indiameter. In some cases, C2A-C2B also assembled into long filaments.Oligomerization, but not membrane binding, was disruptedby neutralization of two lysine residues (K326,327) within the C2Bdomain of syt. These data indicate that Ca21 first drives C2AC2Bzmembraneinteractions, resulting in conformational changesthat trigger a subsequent C2B-mediated oligomerization step.Ca21-mediated rearrangements between syt subunits may regulatethe opening or dilation kinetics of fusion pores or may play arole in endocytosis after fusion.