The Sec1 family of proteins is thought to function in both non-neuronal and neuronal secretion, although the precise role of this protein family has not been defined. Here, we study the function of ROP, the Drosophila Sec1 homolog, in neurotransmitter release. Electrophysiological analyses of transgenic lines overexpressing ROP and syntaxin, a presynaptic membrane protein, indicate that ROP interacts with syntaxin in vivo. Characterization of four point mutations in ROP shows that they fall into two phenotypic classes. Two mutations cause a dramatic reduction in both evoked and spontaneous neurotransmitter release. In contrast, the other two mutations reveal an increase in evoked neurotransmission. Our data further show that neurotransmission is highly sensitive to the levels of ROP function. Studies on heterozygote animals indicate that half the amount of wild-type ROP results in a dramatic decrease in evoked and spontaneous exocytosis. Taken together, these results suggest that ROP interacts with syntaxin in vivo and is a rate-limiting regulator of exocytosis that performs both positive and inhibitory functions in neurotransmission.