Biochemical studies suggest that syntaxin 1A participates in multiple protein-protein interactions in the synaptic terminal, but the in vivo significance of these interactions is poorly understood. We used a targeted mutagenesis approach to eliminate specific syntaxin binding interactions and demonstrate that Drosophila syntaxin 1A plays multiple regulatory roles in neurotransmission in vivo. Syntaxin mutations that eliminate ROP/Munc-18 binding display increased neurotransmitter release, suggesting that ROP inhibits neurosecretion through its interaction with syntaxin. Syntaxin mutations that block Ca2+ channel binding also cause an increase in neurotransmitter release, suggesting that syntaxin normally functions in inhibiting Ca2+ channel opening. Additionally, we identify and characterize a syntaxin Ca2+ effector domain, which may spatially organize the Ca2+ channel, cysteine string protein, and synaptotagmin for effective excitation-secretion coupling in the presynaptic terminal.