Phosphatidylinositol 5-Phosphate (PtdIns5P) is a member of the Phosphoinositide (PI) family of phospholipids, which are intimately involved in many aspects of cellular function. PtdIns5P constitutes approximately 1-5% of total cellular PIs, though its levels have been found to fluctuate in response to certain stimuli and during the cell cycle. Levels of PtdIns5P have been shown to increase after thrombin stimulation in human platelets (Morris et al., 2000), during osmotic stress in mammalian and in plant cells (Meijer et al., 2001), or after insulin stimulation of 3T3-L1 adipocytes. A pool of PtdIns5P in the nucleus of murine erythroleukemia cells is increased 20-fold during the G1 cell cycle phase, and also increased 4-fold following UV irradiation (Jones et al., 2006). Additionally PtdIns5P levels have been shown to decrease in HeLa cells after histamine stimulation (Roberts et al., 2005). PtdIns5P is the least well understood member of the PI family, and its physiological function is presently obscure. Recent evidence suggests that it may act as a transducer of stress signalling, via its interaction with Inhibitor of growth protein-2 (ING2) and p53 regulation (Jones et al., 2006). Although other members of the PI family are well characterised, little is currently known about the mechanisms of synthesis or breakdown of PtdIns5P. It can be produced in vitro via D5 phosphorylation of PtdIns by Type I PIPKins (Phosphatidylinositol Phosphate Kinases) and the Type III PIPKin, PIKfyve. An alternative route is via D3 dephosporylation of PtdIns(3,5)P2 by members of the myotubularin family, or by D4 dephosporylation of PtdIns(4,5)P2 by the S. flexneri effector IpgD and by two recently identified human enzymes, PtdIns(4,5)P2 4-Phospatase Type I and Type II (Ungewickell et al., 2005). PtdIns5P is the preferred substrate of the Type II PIPKins which catalyse its phosphorylation at the D4 position, producing PtdIns(4,5)P2, and recent evidence suggests that the physiological role of these enzymes may be to remove PtdIns5P (Jones et al., 2006). In this study, we demonstrate that PtdIns5P levels are increased in HeLa cells following short-term treatment with Pervanadate. To elucidate the cellular mechanisms involved in the PtdIn5P synthesis we have used siRNA knockdown and overexpression of enzymes suspected to be involved in PtdIns5P regulation. The effect of these on resting PtdIns5P and pervandate elevated PtdIns5P levels, determined by specific PtdIns5P mass assay, are reported.