Thrombin stimulation of isolated rabbit ventricular myocytes activates a membrane-associated, Ca²⁺-independent phospholipase A₂ (iPLA₂) that selectively hydrolyzes plasmalogen phospholipids and results in increased production of arachidonic acid and lysoplasmenylcholine (LPlsCho). To determine whether MAP kinase regulates myocardial iPLA₂ activity, we isolated ventricular myocytes from rabbit heart by collagenase digestion and pretreated with MAP kinase inhibitors prior to thrombin stimulation. Pretreatment with PD 98059 to inhibit p42/44 MAP kinase or SB 203580 to inhibit p38 MAP kinase had no significant effect on thrombin-stimulated membrane-associated iPLA₂ activity. Thrombin stimulation resulted in significant increases in both p42/44 and p38 MAP kinase activity after 2 minutes. Pretreatment with the iPLA₂ selective inhibitor bromoenol lactone completely inhibited thrombin-stimulated MAP kinase activity, suggesting that activation of MAP kinases was dependent upon iPLA₂ activation. Ventricular myocyte MAP kinase activity was increased by incubation of the myocytes with lysoplasmenylcholine, a metabolite produced by iPLA₂-catalyzed membrane plasmalogen phospholipid hydrolysis. Taken together, these data suggest that activation of MAP kinases is downstream of and dependent upon iPLA₂ activation in thrombin-stimulated rabbit ventricular myocytes.