In primary cultures of cerebellar granule cells, glutamate, aspartate, and N-methyl-D-aspartate (NMDA) induced a dose-dependent release of [3H]arachidonic acid ([3H]AA) which was selective for these agonists and was inhibited by NMDA receptor antagonists. The agonist-induced [3H]AA release was reduced by quinacrine at concentrations that inhibited phospholipase A2 (PLA2) but affected neither the activity of phospholipase C (PLC) nor the hydrolysis of phosphoinositides induced by glutamate or quisqualate. Thus, the increased formation of AA was due to the receptor-mediated activation of PLA2 rather than to the action of PLC followed by diacylglycerol lipase. The receptor-mediated [3H]AA release was dependent on the presence of extracellular Ca2+ and was mimicked by the Ca2+ ionophore ionomycin. Pretreatment of granule cells with either pertussis or cholera toxin failed to inhibit the receptor-mediated [3H]AA release. Hence, in cerebellar granule cells, the stimulation of NMDA-sensitive glutamate receptors leads to the activation of PLA2 that is mediated by Ca2+ ions entering through the cationic channels functioning as effectors of NMDA receptors. A coupling through a toxin-sensitive GTP-binding protein can be excluded.