The appropriate functioning of the endosomal system is an essential aspect of cell biology, intimately linked to cell-cell signalling and growth regulation. One component of this system, the late endosomal multivesicular body (MVB), is characterised by its many intraluminal vesicles (ILVs), whose formation is dependent on the evolutionarily-conserved Endosomal Sorting Complexes Required for Transport (ESCRT) 0-III. Although some functions of ILVs are yet to be fully elucidated, MVBs can fuse to the plasma membrane, releasing their ILVs as exosomes (nano-sized extracellular vesicles that transmit multiple signals between cells). ILVs are also thought to prevent excessive receptor-ligand mediated signalling and can be degraded following MVB fusion to lysosomes. We are studying the roles of ILVs in vivo in secondary cells of the Drosophila male accessory gland and aim to translate these findings into human systems. These cells have extraordinarily large (5-10 µm diameter) endosomes, allowing us to readily visualise their diversity, abundance and intraluminal contents. We utilise a combination of molecular genetic approaches and advanced microscopy techniques, including super-resolution 3D structured illumination microscopy (SIM), electron and time-lapse wide-field microscopy. Using 3D-SIM we visualise and resolve ILV populations in living cells for the first time. We observe that ILVs carrying exosome markers are formed within the late endosomal MVBs, as expected, but also within recycling endosomes. These two types of ILV are loaded with different cargos, and secreted via different signalling-dependent mechanisms. We provide evidence that ESCRT complexes localise to and are required for the biogenesis of ILVs in both late and recycling endosomes. We show that human cancer cells also form at least two exosome subtypes in the equivalent compartments, suggesting these cell biological processes are evolutionarily conserved. Functional characterisation reveals at least two previously unsuspected roles for the different ILV populations. We show that late endosomal ILVs carry the v-ATPase proton pump, which sets up acidic microdomains around their exterior, which appears to be required for acidification of this compartment. In addition, ILVs in recycling endosomes play an essential role in the maturation of the dense-core compartments involved in regulated secretion. Our studies therefore reveal multiple new sub-compartmental functions for ILVs and ESCRTs, and provide key evidence to help explain the diversity of exosomes and their biological functions.