The lipid mediators generated by phospholipases A2 (PLA2), free arachidonic acid (AA), eicosanoids, and platelet‐activating factor, modulate neuronal activity; when overproduced, some of them become potent neurotoxins. We have shown, using primary cortical neuron cultures, that glutamate and secretory PLA2 (sPLA2) from bee venom (bv sPLA2) and Taipan snake venom (OS2) elicit synergy in inducing neuronal cell death. Low concentrations of sPLA2 are selective ligands of cell‐surface sPLA2 receptors. We investigated which neuronal arachidonoyl phospholipids are targeted by glutamate‐activated cytosolic calcium‐dependent PLA2 (cPLA2) and by sPLA2. Treatment of 3H‐AA‐labeled cortical neurons with mildly toxic concentrations of sPLA2 (25 ng/ml, 1.78 nM) for 45 min resulted in a two‐ to threefold higher loss of 3H‐AA from phosphatidylcholine (PC) than from phosphatidylethanolamine (PE) and in minor changes in other phospholipids. A similar profile, although of greater magnitude, was observed 20 hr posttreatment. Glutamate (80 μM) induced much less mobilization of 3H‐AA than did sPLA2 and resulted in a threefold greater degradation of 3H‐AA PE than of 3H‐AA PC by 20 hr posttreatment. Combining sPLA2 and glutamate resulted in a greater degradation of PC and PE, and the N‐methyl‐D‐aspartate receptor antagonist MK‐801 only blocked glutamate effects. Thus, activation of the arachidonate cascade induced by glutamate and sPLA2 under experimental conditions that lead to neuronal cell death involves the hydrolysis of different (perhaps partially overlapping) cellular phospholipid pools. © 2002 Wiley‐Liss, Inc.