One common feature of all eukaryotic cells is the presence of various specialized organelles, separated by membranes, which necessitates a coordinated trafficking of materials between these subcellular membrane-bound compartments. Especially neurons, with their long lifespan, polarized and often complex morphology, as well as their specialized functions, have particular requirements for membrane trafficking. Not surprisingly, membrane trafficking is involved in all aspects of neuronal development, function, and long-term maintenance. The evolutionary conserved family of small Rab GTPases functions as key regulator of coordinated vesicular trafficking in the endomembrane system. Expression profiling efforts revealed that in Drosophila half of all Rab GTPases are enriched in or specific to the nervous system, in humans it is one-third. However, the exact functions of the majority of nervous system-enriched Rab proteins are still unknown. Thus, studying the individual roles of those Rab GTPases more closely provides a great opportunity to gain more insight into the membrane trafficking networks in neurons. Ultimately, this will surely contribute to the understanding of what keeps neurons and in particular synapses healthy and functional over extended periods of time. In the nervous system, Rab GTPases and the membrane trafficking events these mediate have been widely associated with many neurodegenerative diseases. However, the established relations are often more correlatively than causatively linked, as discussed in Manuscript 1. Regarding the importance of an intact intracellular trafficking machinery for the development as well as neuronal function and maintenance, I primarily focused on the systematic functional analysis of nervous system-enriched Rab GTPases in Drosophila during my doctoral work. Previously, no systematic rab mutant characterization in any multicellular organism had been performed. The analysis, presented in Manuscript 2, revealed that the homozygous mutants of all nervous system-enriched Rab GTPases, raised under laboratory conditions, are viable and fertile, whereas, null mutants of ubiquitously expressed Rabs are all lethal under homozygosity. Thus, suggesting that Rab proteins, with high expression in the nervous system, serve more modulatory, specialized functions which are not essential for the survival of the organism. Further, we could show that all viable rab mutants differentially affect the development or neuronal function under variable, more challenging environmental conditions, such as temperature and light. This highlights the evolved robustness of developmental processes and nervous system function towards varying conditions. Additionally, during the in-depth functional analysis of nervous system-enriched Rab26, we revealed a stimulus-dependent role in the trafficking of the single acetylcholine receptor subunit Dα4 at cholinergic synapses of outer photoreceptors. However, we could not verify a role for Rab26 in the autophagic turnover of synaptic vesicles in neurons. Additional assays, such as the RUSH system, can be useful to support functional analyses. While this acute, synchronous release system could be established for Rabs in developing photoreceptors and salivary glands, wing imaginal discs proofed to be more sensitive and no working conditions could be established. Using the RUSH assay, different release dynamics with ‘fast’ as well as ‘slow’ releasing Rab GTPases could be identified as shown in Manuscript 3. Further, two nervous system-enriched Rab proteins, namely Rab23 and Rab26, show a clear re-localization from the cell body to the axon terminals, which is in agreement with their predominant synaptic neuropil localization revealed by expression profiling. In conclusion, the findings made during my doctoral work will contribute to a better understanding of the functional requirements of neurons regarding Rab-mediated membrane trafficking. The complete rab null mutant collection as well as the RUSH and LAMA transgenic toolbox provide a strong basis for further investigations of individual Rab functions during development and homeostasis.