Phosphoinositides play an important role in organelle identity by recruiting effector proteins to the host membrane organelle, thus decorating that organelle with molecular identity. Phosphatidylinositol-3,5-bisphos- phate [PtdIns(3,5)P2] is a low-abundance phosphoinositide that predominates in endolysosomes in higher eukaryotes and in the yeast vacuole. Compared to other phosphoinositides such as PtdIns(4,5)P2, our understanding of the regulation and function of PtdIns(3,5)P2 remained rudimentary until more recently. Here, we review many of the recent developments in PtdIns(3,5)P2 function and regulation. PtdIns(3,5)P2 is now known to espouse functions, not only in the regulation of endolysosome morphology, trafficking and acidification, but also in autophagy, signaling mediation in response to stresses and hormonal cues and control of membrane and ion transport. In fact, PtdIns(3,5)P2 misregulation is now linked with several human neuropathologies including Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis. Given the functional versatility of PtdIns(3,5)P2, it is not surprising that regulation of PtdIns(3,5)P2 metabolism is proving rather elaborate. PtdIns(3,5)P2 synthesis and turnover are tightly coupled via a protein complex that includes the Fab1/PIKfyve lipid kinase and its antagonistic Fig4/Sac3 lipid phosphatase. Most interestingly, many PtdIns(3,5)P2 regulators play simultaneous roles in its synthesis and turnover.