Lysosomes are dynamic, terminal organelles of both the endocytic and autophagic pathways which play a role in macromolecule degradation, nutrient sensing, signalling to the cell nucleus, and plasma membrane repair1,2. They are classically described as having an acidic lumen which enables efficient function of their resident acid hydrolases; however many studies indicate heterogeneity of lysosomal pH within individual cells, including lysosomes with a neutral luminal pH. The endocytic delivery of macromolecules from the mammalian cell surface for degradation by lysosomal acid hydrolases requires traffic through early endosomes to late endosomes, followed by transient (kissing) or complete fusions between late endosomes and lysosomes1. Transient or complete fusion results in the formation of endolysosomes3, which are hybrid organelles from which lysosomes are re-formed1,3. Through the use of fluorescent reporters of cathepsin activity and acid phosphatase cytochemistry, combined with immunolocalization of endosomal and lysosomal markers, we have found that acid hydrolase activity occurs predominantly in the endolysosomal compartment4. Thus, endolysosomes are the principal organelles in which acid hydrolase substrates are cleaved. Endolysosomes can be distinguished from re-usable terminal lysosomes by their accumulation of membrane-permeable cathepsin activity reporters and fluorescent acidotropic probes, neither of which accumulate in more neutral terminal lysosomes. These neutral lysosomes contain inactive acid hydrolases that can be detected by immunoelectron microscopy, suggesting that they may function as a re-usable storage compartment for these enzymes. Using live cell microscopy of NRK (normal rat kidney) fibroblasts, we have demonstrated that transient fusion events, resulting in the formation of endolysosomes, precede the onset of acid hydrolase activity4. Thus, kissing may be regarded as nucleating acid hydrolase activity. By means of sucrose and invertase uptake experiments, we have also shown that acid hydrolase-active endolysosomes and acid hydrolase-inactive, terminal lysosomes exist in dynamic equilibrium. Thus, fluid phase endocytosis of sucrose by NRK cells resulted in the accumulation of sucrose-laden osmotically swollen endolysosomes (sucrosomes) and depletion of the terminal lysosome pool within the cells. These cells were subsequently allowed to endocytose invertase, enabling the hydrolysis of sucrose, which resulted in tubulation from the sucrosomes and eventual reformation of the normal proportion of re-usable terminal lysosomes4. The sucrose treatment of the NRK cells did not cause an increae in autophagosomes, nor increased translocation of transcription factor EB to the nucleus4. Many questions remain about the lysosome-endosome fusion and regeneration cycle. These include, but are not restricted to: whether the machinery of fusion determined mainly from cell-free assays is sufficient to explain fusion in living cells. We have recent evidence for SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) compensation in cells in which individual SNARES are knocked out; the relationship between the transient pores required for kissing and the expansion of pores to allow full fusion – we suggest a role for tether clearance to achieve full fusion; the regulation of luminal acidity of organelles in the lysosome-endosome fusion and regeneration cycle. The acidic lumen of these organelles requires the proton pumping V-ATPase activity and cation channels and a Cl-/H+ antiporter have been implicated in the necessary charge compensation. Alteration of luminal acidity may involve regulation of any of these components or other factors such as passive(leak) permeability to protons. Using fluorescent protein-tagged constructs of V-ATPase subunits we are currently exploring the dynamics of the V-ATPase during lysosome reformation in the sucrosome/invertase cell model described above; the molecular machinery of reformation of re-usable terminal storage lysosome reformation from endolysosomes and whether it is the same or different to that reported for lysosome reformation from autolysosomes5; the relationship between the molecular machineries for lysosome reformation and subcellular localization. This is relevant because others have reported that, in several cell types, peripheral lysosomes are less acidic than juxtanuclear lysosomes, as a consequence of their subcellular location6.