There is persuasive evidence that Ca2+ sensor at the nerve terminal is synaptotagmin, an integral protein of the synaptic vesicle membrane. In response to Ca2+ binding, synaptotagmin interacts via its cytoplasmic domains C2A and C2B both with negatively charged phospholipids, and with SNAREs. It is now accepted that both C2A and C2B domains undergo Ca2+- triggered associations with negatively charged phospholipids. This association is intimate, and involves the penetration of the Ca2+-binding regions of the C2 domains into the lipid bilayer. In the present study, we imaged the binding of synaptotagmin to supported lipid bilayers under fluid using atomic force microscopy. This technique provides the opportunity to study the behaviour of synaptotagmin at the single-molecule level under near-physiological conditions. We show that C2AB binds to lipid bilayers containing negatively charged phospholipids in a Ca2+- dependent manner. Bound synaptotagmin is oligomeric, but does not seem to adopt a single stoichiometry. Intriguingly, the binding of C2AB induces the formation of stable indentations in the bilayers, suggesting that it is able to significantly perturb the bilayer structure. The possible significance of this perturbation with respect to Ca2+-triggered membrane fusion is discussed.