Synaptotagmin I is the Ca(2+) sensor for fast, synchronous release of neurotransmitter; however, the molecular interactions that couple Ca(2+) binding to membrane fusion remain unclear. The structure of synaptotagmin is dominated by two C(2) domains that interact with negatively charged membranes after binding Ca(2+). In vitro work has implicated a conserved basic residue at the tip of loop 3 of the Ca(2+)-binding pocket in both C(2) domains in coordinating this electrostatic interaction with anionic membranes. Although results from cultured cells suggest that the basic residue of the C(2)A domain is functionally significant, such studies provide contradictory results regarding the importance of the C(2)B basic residue during vesicle fusion. To directly test the functional significance of each of these residues at an intact synapse in vivo, we neutralized either the C(2)A or the C(2)B basic residue and assessed synaptic transmission at the Drosophila neuromuscular junction. The conserved basic residues at the tip of the Ca(2+)-binding pocket of both the C(2)A and C(2)B domains mediate Ca(2+)-dependent interactions with anionic membranes and are required for efficient evoked transmitter release. Our results directly support the hypothesis that the interactions between synaptotagmin and the presynaptic membrane, which are mediated by the basic residues at the tip of both the C(2)A and C(2)B Ca(2+)-binding pockets, are critical for coupling Ca(2+) influx with vesicle fusion during synaptic transmission in vivo. Our model for synaptotagmin's direct role in coupling Ca(2+) binding to vesicle fusion incorporates this finding with results from multiple in vitro and in vivo studies.