Aims: Ethanol affects not only the cytoskeletal organization and activity, but also intracellular trafficking in neurons in the primary culture. Polyphosphoinositide (PPIn) are essential regulators of many important cell functions, including those mentioned, cytoskeleton integrity and intracellular vesicle trafficking. Since information about the effect of chronic ethanol exposure on PPIn metabolism in neurons is scarce, this study analysed the effect of this treatment on three of these phospholipids. Methods: Phosphatidylinositol (PtdIns) levels as well as the activity and/or levels of enzymes involved in their metabolism were analysed in neurons chronically exposed to ethanol. The levels of phospholipases C and D, and phosphatidylethanol formation were also assessed. The consequence of the possible alterations in the levels of PtdIns on the Golgi complex (GC) was also analysed. Results: We show that phosphatidylinositol (4,5)-bisphosphate and phosphatidylinositol (3,4,5)-trisphosphate levels, both involved in the control of intracellular trafficking and cytoskeleton organization, decrease in ethanol-exposed hippocampal neurons. In contrast, several kinases that participate in the metabolism of these phospholipids, and the level and/or activity of phospholipases C and D, increase in cells after ethanol exposure. Ethanol also promotes phosphatidylethanol formation in neurons, which can result in the suppression of phosphatidic acid synthesis and, therefore, in PPIn biosynthesis. This treatment also lowers the phosphatidylinositol 4-phosphate levels, the main PPIn in the GC, with alterations in their morphology and in the levels of some of the proteins involved in structure maintenance. Conclusions: The deregulation of the metabolism of PtdIns may underlie the ethanol-induced alterations on different neuronal processes, including intracellular trafficking and cytoskeletal integrity.