Synaptic communication requires the controlled release of synaptic vesicles from presynaptic axon terminals. Release efficacy is regulated by the many proteins that comprise the presynaptic release apparatus, including Ca2+channels and proteins that influence Ca2+channel accumulation at release sites. Here we identifyDrosophilaRIM (Rab3 interacting molecule) and demonstrate that it localizes to active zones at the larval neuromuscular junction. InDrosophilaRIM mutants, there is a large decrease in evoked synaptic transmission because of a significant reduction in both the clustering of Ca2+channels and the size of the readily releasable pool of synaptic vesicles at active zones. Hence, RIM plays an evolutionarily conserved role in regulating synaptic calcium channel localization and readily releasable pool size. Because RIM has traditionally been studied as an effector of Rab3 function, we investigate whether RIM is involved in the newly identified function of Rab3 in the distribution of presynaptic release machinery components across release sites. Bruchpilot (Brp), an essential component of the active zone cytomatrix T bar, is unaffected byRIMdisruption, indicating that Brp localization and distribution across active zones does not require wild-type RIM. In addition, larvae containing mutations in bothRIMandrab3have reduced Ca2+channel levels and a Brp distribution that is very similar to that of therab3single mutant, indicating that RIM functions to regulate Ca2+channel accumulation but is not a Rab3 effector for release machinery distribution across release sites.