Synaptotagmin I is a synaptic vesicle protein postulated to mediate vesicle docking, vesicle recycling, and the Ca2+ sensing required to trigger vesicle fusion. Analysis of synaptotagmin I knockouts (sytINULL mutants) in both Drosophila and mice led to these hypotheses. Although much research on the mechanisms of synaptic transmission in Drosophila is performed at the third instar neuromuscular junction, the ultrastructure of this synapse has never been analyzed in sytINULL mutants. Here we report severe synaptic vesicle depletion, an accumulation of large vesicles, and decreased vesicle docking at sytINULL third instar neuromuscular junctions. Mutations in synaptotagmin I's C2B Ca2+-binding motif nearly abolish synaptic transmission and decrease the apparent Ca2+ affinity of neurotransmitter release. Although this result is consistent with disruption of the Ca2+ sensor, synaptic vesicle depletion and/or redistribution away from the site of Ca2+ influx could produce a similar phenotype. To address this question, we examined vesicle distributions at neuromuscular junctions from third instar C2B Ca2+-binding motif mutants and transgenic wild-type controls. The number of docked vesicles and the overall number of synaptic vesicles in the vicinity of active zones was unchanged in the mutants. We conclude that the near elimination of synaptic transmission and the decrease in the Ca2+ affinity of release observed in C2B Ca2+-binding motif mutants is not due to altered synaptic vesicle distribution but rather is a direct result of disrupting synaptotagmin I's ability to bind Ca2+. Thus, Ca2+ binding by the C2B domain mediates a postdocking step in fusion. J. Comp. Neurol. 496:1–12, 2006. © 2006 Wiley-Liss, Inc.