AbstractRIM-Binding Protein 2 (RIM-BP2) is a multi-domain protein of the presynaptic active zone (AZ). By binding to Rab-interacting protein (RIM), bassoon and voltage-gated Ca²⁺ channels (CaV), it is considered to be a central organizer of the topography of CaVand release sites of synaptic vesicles (SVs) at the AZ. Here, we investigated the role of RIM-BP2 at the endbulb of Held synapse of auditory nerve fibers with bushy cells of the cochlear nucleus, a fast relay of the auditory pathway with high release probability. Disruption of RIM-BP2 lowered release probability altering short-term plasticity and reduced evoked excitatory postsynaptic currents (EPSCs). Analysis of SV pool dynamics during high frequency train stimulation indicated a reduction of SVs with high release probability but an overall normal size of the readily releasable SV pool (RRP). The Ca2+-dependent fast component of SV replenishment after RRP depletion was slowed. Ultrastructural analysis by super-resolution light and electron microscopy revealed an impaired topography of presynaptic CaVand a reduction of docked and membrane-proximal SVs at the AZ. We conclude that RIM-BP2 organizes the topography of CaV, and promotes SV tethering and docking. This way RIM-BP2 is critical for establishing a high initial release probability as required to reliably signal sound onset information that we found to be degraded in bushy cells of RIM-BP2-deficient micein vivo.Significance StatementRIM-binding proteins (RIM-BPs) are key organizers of the active zone (AZ). Using a multidisciplinary approach to the calyceal endbulb of Held synapse that transmit auditory information at rates of up to hundreds of Hertz with sub-millisecond precision we demonstrate a requirement for RIM-BP2 for normal auditory signaling. Endbulb synapses lacking RIM-BP2 show a reduced release probability despite normal whole-terminal Ca2+influx and abundance of the key priming protein Munc13-1, a reduced rate of SV replenishment, as well as an altered topography of CaV2.1 Ca2+channels, and fewer docked and membrane proximal synaptic vesicles. This hampers transmission of sound onset information likely affecting downstream neural computations such as of sound localization.