Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)) is the most recently discovered PtdInsP(2) isomer. It is likely that PtdIns(3,5)P(2) is ubiquitous to eukaryotes, and that it performs a number of important cellular functions, including vacuolar homeostasis, retrograde trafficking from the vacuole, and protein sorting at the multivesicular body. This review describes the metabolism of PtdIns(3,5)P(2) and discusses the potential functions for this lipid.