It is usually assumed that neurotransmitter is released in an all or none manner from synaptic vesicles. However, recently it was suggested (Wang et al., 2003 Nature 424:943-7), that in large neuroendocrine vesicles, the tandem calcium-binding C2 domains C₂A and C₂B of the synaptic vesicle protein synaptotagmin 1 (syt1) control neurotransmitter release through fusion pores. To test for fusion pore effects on release from small vesicles we studied currents at neuromuscular junctions in syt1 Drosophila mutants, using the time course of evoked postsynaptic quantal responses as readout for the time course of glutamate efflux from vesicles. While disrupting Ca²⁺-binding in the syt1 C₂A domain (syt1 D229N) did not alter quantal decay (6,3 ± 0,3 ms) disrupting Ca²⁺-binding in the syt1 C₂B domain (syt1 D3,4N) accelerated quantal decay (5,2 ± 0,4 ms). Furthermore mutating the syt1 C₂B polylysine motif (syt1 KQ; 4,9 ± 0,3 ms) also accelerated quantal decay relative to controls (6,4 ± 0,3 ms). These results are consistent with the interpretation that syt1 is a key element in fusion pore regulated transmitter discharge from synaptic vesicles in neurons. The molecular mechanism which allows syt1 to control fusion pores remains to be interpreted. In view of the many binding partners and putative functions of syt1 several scenarios are conceivable. Attractive may be the interpretation that syt1 oligomerisation either slows or prevents fusion pore dilation.