Transmitter release from active zones of presynaptic nerve terminals is the most important means of fast information transfer between neurons in the brain. In recent years, significant progress in our understanding of the molecular mechanisms of presynaptic active zone function has been achieved by creating and analyzing k.o. mouse models deficient for various presynaptic proteins. However, important aspects of the coupling between Ca²⁺ channels and readily-releasable vesicles, as well as of the Ca²⁺ triggering function in transmitter release have remained obscure, maybe because of the inaccessibility of many small synapses to direct presynaptic electrophysiology. The calyx of Held is a large synapse in the auditory brainstem of mammals at which precise control of the nerve terminal [Ca²⁺]_i concentration can be achieved (Schneggenburger & Neher 2005, Curr. Op. Neurobiol.). However, molecular perturbation of this large brainstem synapse has been difficult, since many k.o. mouse models of presynaptic proteins are perinatally lethal, and since rescue of presynaptic function at the calyx of Held in a k.o. mouse model has not yet been obtained. Here, in a first study on the role of Synaptotagmin in Ca²⁺-triggered vesicle fusion, we have established a functional rescue of Ca²⁺ triggered release at the calyx of Held in Synaptotagmin-2 k.o. mice, by using adenovirus-mediated overexpression of Syt-2. This has allowed us to perform a structure-function analysis of Syt-2 function under direct control of presynaptic [Ca²⁺]_i. In a second study, we have established a tissue-specific k.o. of all known RIM 1/2 isoforms (Rab3a-interacting molecule) at the calyx of Held, using recently generated floxed mouse lines for RIM1 (Kaeser et al. 2008, J. Neuroscience) and RIM2, crossed with an auditory-brainstem specific Cre mouse line. This approach avoids perinatal lethality, and promises to yield new insights into the roles of RIM's in docking and priming of readily-releasable vesicles, and in targeting Ca²⁺ channels to the active zone.