Cytosolic phospholipase A(2) (cPLA(2)) stimulates the hydrolysis of sn-2 ester bond in membrane phospholipids releasing arachidonic acid (AA) and lysophospholipids. The present study examined the effect of methylmercury (MeHg) on cPLA(2) activation and AA release in primary cultures of neonatal rat cerebral astrocytes. Astrocytes were preloaded overnight at 37 degrees C with 3H-AA to metabolically label phospholipids. The effect of MeHg on the activation of cPLA(2) was measured by the release of 3H-AA from astrocytes over 120 min. MeHg (5 microM) caused a significant increase in AA release at 10, 30, 60, and 120 min, whereas 2.5 microM MeHg significantly increased AA release only at 120 min. MeHg-induced increase in 3H-AA release was due to cPLA(2) activation, since arachidonyl trifluoromethyl ketone (AACOCF(3)), a selective inhibitor of cPLA(2), completely abolished MeHg's effect. Consistent with these observations, MeHg (5.0 and 10.0 microM) increased cPLA(2) mRNA (6 h) and cPLA(2) protein expression (5.0 and 10.0 microM; 24 h). The time-course of these effects suggests an immediate direct or indirect effect of MeHg on cPLA(2) activation and 3H-AA release as well as a long-term effect involving the induction of cPLA(2). Thin layer chromatographic analysis of 3H-AA-labeled phospholipids showed that MeHg-stimulated astrocyte 3H-AA release was not due to increased incorporation of 3H-AA into the putative substrates of cPLA(2). These results invoke cPLA(2) as a putative target for MeHg toxicity, and support the notion that cPLA(2)-stimulated hydrolysis and release of AA play a critical role in MeHg-induced neurotoxicity.