Lysosomes function as the terminal degradative compartment of a cell’s endocytic and autophagic pathways, and as a multifunctional signalling hub integrating the cell’s response to nutrient status and growth factor/hormone signalling. They have long been known as acidic organelles although it is now clear that the luminal pH in the lysosomal compartment can vary between <pH4.5 and close to neutral pH7. Regulation of the luminal pH in continuously fed mammalian cells is poorly understood. In this study from the Luzio lab, the reversible assembly/disassembly of the proton pumping V-ATPase was demonstrated when acidic endolysosomes are formed by kissing and fusion of late endosomes with lysosomes and during the subsequent reformation of lysosomes. Using confocal microscopy and subcellular fractionation of cells stably expressing fluorescently tagged proteins, net recruitment of the V-ATPase V1 subcomplex during acidic endolysosome formation was observed and loss during neutral lysosome reformation, with a similar time course for loss of the small GTPase RAB7A. Live cell microscopy additionally showed the dynamic equilibrium and rapid exchange between cytosolic and membrane-bound pools of a V1 subcomplex protein. The paper shows that reversible V-ATPase assembly/disassembly plays a key role in regulating endolysosomal/lysosomal pH in continuously fed cells. It also strongly suggests that a RAB7A effector(s) is required for V-ATPase assembly and speculates that one such effector may be DMXL1, a known interactor of RAB7A. This protein was recently shown, by the Weekes group at CIMR, to be degraded following human cytomegalovirus (HCMV) infection, resulting in inhibition of lysosome acidification.