At least four forms of synaptotagmin are expressed in neurons. Of these, synaptotagmin I has an essential function in mediating Ca²⁺-triggered neurotransmitter release at hippocampal synapses, but the functional implications of multiple synaptotagmins are unknown. Synaptotagmins I-III exhibit a strikingly differential distribution between synapses, with most neurons coexpressing either synaptotagmins I or II with III. Synaptotagmin IV is present uniformly throughout the brain at low levels. Synaptotagmins III and IV are both coexpressed with synaptotagmin I in hippocampal synapses, suggesting that these synaptotagmins are not functionally redundant. The first C₂ domains of synaptotagmins I-III exhibit similar Ca²⁺ affinities in phospholipid-binding assays, whereas that of synaptotagmin IV is unable to bind Ca²⁺. All synaptotagmins tested bind the clathrin-adaptor protein AP-2 with high affinity. Our results suggest that different synaptotagmins serve distinct but overlapping functions in neuronal membrane traffic, with synaptotagmins I and II representing alternative Ca²⁺ sensors in exocytosis and all synaptotagmins functioning as AP-2 receptors in endocytosis.