Synaptic vesicle exocytosis is regulated by several proteins localized at the active zone of presynaptic terminals. Munc13-1 is a multi-domain active zone protein which redundantly interacts with other active zone proteins. We previously found that Munc13-1 forms supramolecular nanoassemblies that correspond to the sites of synaptic vesicle exocytosis. Here, we aimed to clarify a more detailed molecular mechanism of Munc13-1 in the formation of synaptic vesicle release sites by intervening in the interactions between Munc13-1 and other active zone proteins. For this purpose, we acquired a series of isolated functional domains which directly bind to Munc13-1, from active zone proteins including RIM, CAST, RIM-BP, and Piccolo. Among these domains, we found that the expression of Zn²⁺ finger domain of RIM (RIM-ZF), which binds to C₂A domain of Munc13-1, caused a significant decrease in neurotransmitter release, although a previous study showed that the RIM-ZF partially rescues suppressed neurotransmitter release in RIM knock-out neurons. Furthermore, quantitative immunocytochemical analysis with super-resolution microscopy imaging revealed that the expression of RIM-ZF selectively reduced the amounts of Munc13-1 molecules at the active zones. Thus, our results suggest that a direct interaction of RIM-ZF with Munc13-1 itself is incomplete for the appropriate formation of synaptic vesicle release sites, and that cross-linkage of RIM-ZF to the other domains of RIM is necessary for the precise positioning of Munc13-1 at the active zone.