C 2-domains are widespread protein modules with diverse Ca 2+-regulatory functions. Although multiple Ca 2+ ions are known to bind at the tip of several C 2-domains, the exact number of Ca 2+-binding sites and their functional relevance are unknown. The first C 2-domain of synaptotagmin I is believed to play a key role in neurotransmitter release via its Ca 2+-dependent interactions with syntaxin and phospholipids. We have studied the Ca 2+-binding mode of this C 2-domain as a prototypical C 2-domain using NMR spectroscopy and site-directed mutagenesis. The C 2-domain is an elliptical module composed of a β-sandwich with a long axis of 50 Å. Our results reveal that the C 2-domain binds three Ca 2+ ions in a tight cluster spanning only 6 Å at the tip of the module. The Ca 2+-binding region is formed by two loops whose conformation is stabilized by Ca 2+ binding. Binding involves one serine and five aspartate residues that are conserved in numerous C 2-domains. All three Ca 2+ ions are required for the interactions of the C 2-domain with syntaxin and phospholipids. These results support an electrostatic switch model for C 2-domain function whereby the β-sheets of the domain provide a fixed scaffold for the Ca 2+-binding loops, and whereby interactions with target molecules are triggered by a Ca 2+-induced switch in electrostatic potential.