Treatment of cells with agents that stimulate the release of arachldonic acid causes increased serine phosphorylation and activation of cytosolic phospholipase A2 (cPLA*). Here we report that cPLAz is a substrate for mitogen-activated protein (MAP) kinase. Moreover, phosphorylation by MAP kinase increases the enzymatic activity of cPLA2. The site of cPLAz phosphorylatlon by MAP kinase, Ser-505, is identical to the major site of cPLA2 phosphorylation observed in phorbol ester-treated cells. Replacement of Ser-505 with Ala resulted in a mutant cPLA2 that is not a substrate for MAP kinase and causes little or no enhanced agonist-stimulated arachidonate release from intact cells. Taken together, these data indicate that MAP kinase mediates, at least in part, the agonist-induced activation of cPLA2.