The mammalian homolog of the yeast Sec1p, n-Sec1/Munc-18 has been demonstrated to bind the presynaptic membrane protein syntaxin, a putative synaptic vesicle docking protein. To determine the role of n-Sec1/Munc-18 in neurotransmitter release in vivo, we have overexpressed the Drosophila homolog, rop, in third instar larvae and measured the electrophysiological consequences at the neuromuscular junction. A 3- to 5-fold induction of the rop protein causes a dramatic decrease in neurotransmitter release, suggesting rop may restrict the ability of vesicles to dock or of docked vesicles to fuse. Consistent with this hypothesis, rop overexpression also reduces the number of spontaneous vesicle fusions by more than 50%, and repetitive stimulation results in significant decreases in evoked responses similar to those observed in rab3a mutant mice. However, rop overexpression does not alter significantly the Ca2+ dependence of neurotransmitter release. We propose that the Drosophila n-Sec1/Munc-18 homolog plays a negative role in neurotransmitter release in vivo, in addition to its previously identified positive function, possibly by modulation of docking of synaptic vesicles or activation of a pre-fusion complex at the active zone.