Fast neurotransmitter release is essential for neuron-neuron communication and is initiated by the opening of voltage-gated Ca2+ channels close to docked vesicles at the presynaptic active zone. The high concentration of Ca2+ channels at the active zone is important to secure fast transmitter release. However, the mechanism that enriches Ca2+ channels at active zones is largely unknown, maybe because of the limited accessibility of most model synapses to direct measurements of Ca2+ signaling in the nerve terminal. RIM's (Rab3 interacting molecule) are scaffolding proteins in the active zone which interact with several presynaptic proteins. As revealed by previous studies in C. elegans and cultured neurons, RIM proteins affect synaptic transmission. However, their complex functions have not been clearly dissected, because direct access to the presynaptic nerve terminal has so far not been possible in synapses that are amenable to genetic manipulation. Here, we have established a Cre-lox based conditional KO approach at a presynaptically accessible CNS synapse, the calyx of Held. This has allowed us to use presynaptic recordings and Ca2+ uncaging, as well as electron microscopic analyses of synapses which have developed in vivo, to directly study the presynaptic functions of RIM proteins. We discovered three major functions of RIM proteins: First, RIM's hold Ca2+ channels in the presynaptic terminals to secure the high Ca2+ channel density at active zones. Removal of RIM proteins leads to a decrease of presynaptic Ca2+ current amplitude (by ∼ 50%) as well as to a decreased immunostaining signal using an antibody against a Ca2+ channel α-subunit (by ∼ 40%). Second, RIM proteins dock vesicles to the active zone and therefore control the size of readily releasable pool. In RIM1/2 cDKO synapses, the number of docked vesicles is reduced by ∼ 80% and the functional pool size is decreased by ∼ 75%. The good correlation of morphological and physiological data identified RIM as the first presynaptic protein in a CNS synapse with a clearly corresponding role in vesicle docking, and priming. Third, RIM proteins increase the release probability and speed up the release kinetics. This is carried out by two new functions: increasing the intrinsic Ca2+ sensitivity of release and tightly coupling docked vesicles to Ca2+ channels. Taken together, this study shows that RIM proteins function as the central organizers to coordinate active zone function: holding the Ca2+ channels in the presynaptic terminal, docking vesicles to the active zones, and regulating the release probability of any given readily-releasable vesicle. All the three functions together secure the fast vesicle release when the AP arrives in the presynaptic terminal.