Phosphatidylinositol 5-phosphate (PtdIns5P) is the least well-characterised member of the phosphoinositide family of essential regulatory phospholipids. PtdIns5P levels are altered within cells in response to a number of stimuli, and evidence is accumulating to suggest that it possesses important functions in cellular signalling. However, the physiological role of this lipid remains imperfectly understood. Previous studies have shown that PtdIns5P is elevated in adipocytes in response to insulin, and microinjection of PtdIns5P into these cells promotes plasma membrane insertion of the insulin-regulated glucose transporter GLUT4 (Sbrissa et al., 2004). This finding suggests a potential role of PtdIns5P as a mediator in insulin-stimulated glucose uptake, a process essential for efficient glucose homeostasis. As approximately 75% of post-prandial glucose absorption occurs into skeletal muscle, it is important to investigate the role of PtdIns5P in the insulin response of this tissue. We have therefore used differentiated myotubes of the rat muscle cell line, L6, to explore the effects of altered PtdIns5P levels on insulin-stimulated glucose uptake. Here we show that insulin stimulates a transient increase of PtdIns5P in L6 myotubes, and that abolition of this increase by overexpression of the PtdIns5P kinase PIP4K-alpha abolishes insulin-stimulated glucose uptake. Interestingly, artificial elevation of PtdIns5P in L6 myotubes increases glucose uptake in the absence of stimulation. This phenomenon appears to result from the activation of PI 3-kinase signalling, as it is abolished by the PI 3-kinase inhibitor wortmannin, and involves activation of the PI 3-kinase effector Akt. Our results are consistent with the idea that insulin-stimulated PtdIns5P production contributes to the robust PI 3-kinase/Akt activation necessary for insulin-stimulated glucose uptake in muscle.