Synaptotagmin I is a synaptic vesicle protein postulated to mediate vesicle docking, vesicle recycling, and the Ca(2+) sensing required to trigger vesicle fusion. Analysis of synaptotagmin I knockouts (sytI(NULL) 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 sytI(NULL) mutants. Here we report severe synaptic vesicle depletion, an accumulation of large vesicles, and decreased vesicle docking at sytI(NULL) third instar neuromuscular junctions. Mutations in synaptotagmin I's C(2)B Ca(2+)-binding motif nearly abolish synaptic transmission and decrease the apparent Ca(2+) affinity of neurotransmitter release. Although this result is consistent with disruption of the Ca(2+) sensor, synaptic vesicle depletion and/or redistribution away from the site of Ca(2+) influx could produce a similar phenotype. To address this question, we examined vesicle distributions at neuromuscular junctions from third instar C(2)B Ca(2+)-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 Ca(2+) affinity of release observed in C(2)B Ca(2+)-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 Ca(2+). Thus, Ca(2+) binding by the C(2)B domain mediates a post-docking step in fusion.