The eicosanoids are centrally involved in the onset and resolution of inflammatory processes. A key enzyme in eicosanoid biosynthesis during inflammation is group IVA phospholipase A₂ (also known as cytosolic phospholipase A_2α, cPLA₂α). This enzyme is responsible for generating free arachidonic acid from membrane phospholipids. cPLA₂α translocates to perinuclear membranes shortly after cell activation, in a process that is governed by the increased availability of intracellular Ca²⁺. However, cPLA₂α also catalyzes membrane phospholipid hydrolysis in response to agonists that do not mobilize intracellular Ca²⁺. How cPLA₂α interacts with membranes under these conditions is a major, still unresolved issue. Here, we report that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] promotes translocation of cPLA₂α to perinuclear membranes of intact cells in a manner that is independent of rises in the intracellular Ca²⁺ concentration. PtdIns(4,5)P₂ anchors the enzyme to perinuclear membranes and allows for a proper interaction with its phospholipid substrate to release arachidonic acid.