Through the endocytic pathway, internalized vacuoles mature, acquiring progressively an acidic pH and eventually ending-up to fuse with lysosomes to form endolysosomes. PIKfyve is a membrane-located lipid kinase found in the endocytic pathway that participates in the maturation of endosomes. It is the only enzyme responsible for the conversion of phosphatidylinositol-3-phosphate PI(3)P to PI(3,5)P2, a low abundance phospholipid located in the membrane of late endosomes and lysosomes. PI(3,5)P2 is a signaling lipid which has been shown to directly interact with and modulate the activity of several lysosomal membrane proteins, including the Ca2+ channel TRPML1 involved in endolysosomal trafficking and function. Pharmacological inhibition of PIKfyve leads to PI(3,5)P2 depletion, which quickly and drastically disrupts the late endosomal/lysosomal trafficking, leading to the generation of oversized vacuoles. A recent study shows that knocking out ClC-7, a lysosomal Cl-/H+ exchanger, provided substantial resistance against endocytic trafficking defects resulting from PIKfyve inhibition, such as vacuole formation (Gayle et al., Blood, 2017). Given that ClC-7 has been suggested to play a role in lysosomal acidification, these results raise the possibility that either PIKfyve or PI(3,5)P2 may regulate ClC-7 activity, in turn modulating the lysosomal pH, and that endosomal/lysosomal pH could be a critical parameter for the fine tuning of the endocytic trafficking. Using ratiometric live measurements of lysosomal pH in intact cultured cells, we first observe that pharmacological inhibition of PIKfyve significantly hyperacidifies lysosomes. Repeating this experiment in cells in which ClC7 is knocked out abolish the pharmacologically-induced lysosomal hyperacidification, providing new evidence for the role of ClC7 as a key regulator of lysosomal pH, and suggesting that PIKfyve/PI(3,5)P2 may inhibit ClC7 activity. Finally, we analyze the relationship between lysosomal pH and vacuole formation and present results indicating that they are uncoupled.