Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P 2 ] is a low-abundance phosphoinositide presumed to be localized to endosomes and lysosomes, where it recruits cytoplasmic peripheral proteins and regulates endolysosome-localized membrane channel activity. Cells lacking PI(3,5)P 2 exhibit lysosomal trafficking defects, and human mutations in the PI(3,5)P 2 -metabolizing enzymes cause lysosome-related diseases. The spatial and temporal dynamics of PI (3,5)P 2 , however, remain unclear due to the lack of a reliable detection method. Of the seven known phosphoinositides, only PI (3,5)P 2 binds, in the low nanomolar range, to a cytoplasmic phosphoinositide-interacting domain (ML1N) to activate late endosome and lysosome (LEL)-localized transient receptor potential Mucolipin 1 (TRPML1) channels. Here, we report the generation and characterization of a PI(3,5)P 2 -specific probe, generated by the fusion of fluorescence tags to the tandem repeats of ML1N. The probe was mainly localized to the membranes of Lamp1-positive compartments, and the localization pattern was dynamically altered by either mutations in the probe, or by genetically or pharmacologically manipulating the cellular levels of PI(3,5)P 2 . Through the use of time-lapse live-cell imaging, we found that the localization of the PI(3,5)P 2 probe was regulated by serum withdrawal/addition, undergoing rapid changes immediately before membrane fusion of two LELs. Our development of a PI(3,5)P 2 -specific probe may facilitate studies of both intracellular signal transduction and membrane trafficking in the endosomes and lysosomes.TRP channel | PIKfyve | vesicle fusion P hosphorylated phosphoinositide lipids are produced on the cytosolic side of cellular lipid bilayer membranes (1, 2). There are seven different known phosphoinositides lipids, which localize to distinct membrane subdomains to regulate organellespecific membrane signaling pathways and membrane-trafficking events (1, 2). One such phosphoinositide lipid is phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ], which is predominantly localized to the plasma membrane (PM). PI(4,5)P 2 serves as a permissive cofactor that is required for the activity of PM channels/transporters, as a precursor for the generation of second messengers, and as a PM recruiter for cytosolic peripheral proteins (3, 4). Likewise, PI(3,4)P 2 and PI(3,4,5)P 3 are transiently produced at the PM to regulate various signaling effectors, such as v-akt-murine thymoma viral oncogene homolog 1 (Akt) (1, 5). Conversely, PI(3)P is primarily found on early endosomes, phagosomes, and autophagosomes to regulate the maturation of these compartments (5, 6).Unlike the aforementioned phosphoinositides, the subcellular localization and functions of PI(3,5)P 2 are poorly understood. PI (3,5)P 2 is proposed to be mainly localized to late endosomes and lysosomes (LELs) (7), and also to early endosomes (8), based on the location of its synthesizing enzyme complex, which in mammalian cells consists of the phosphoinositide kinase, FYVE finger-containing (...