Patients with transfusional iron overload may develop a life-limiting cardiomyopathy. The sensitivity of lipid-metabolizing enzymes to peroxidative injury, as well as the reported effects of arachidonic acid (AA) and metabolites on cardiac rhythm, led us to hypothesize that iron-overloaded cardiomyocytes display alterations in the release of AA and prostaglandins. Neonatal rat ventricular myocytes (NRVMs) cultured for 72 hours in the presence of 80 microgram/mL ferric ammonium citrate displayed an increased rate of AA release, both under resting conditions and after stimulation with agonists such as [Sar(1)]Ang II. Although iron treatment did not affect overall incorporation of [(3)H]AA into NRVM phospholipids, it caused a 2-fold increase in the distribution of precursor in phosphatidylcholine species, with a proportional decrease in phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine. Increased release of AA in iron-overloaded NRVMs was reduced by the diacylglycerol lipase inhibitor RHC80267 but was largely insensitive to inhibitors of phospholipases A(2) and C. Iron-overloaded cardiomyocytes also displayed increased production of eicosanoids and induction of cyclooxygenase-2 after stimulation with interleukin-1alpha. Iron overload enhances AA release and incorporation of AA into phosphatidylcholine, as well as cyclooxygenase-2 induction and eicosanoid production, in NRVMS: The effects of AA and metabolites on cardiomyocyte rhythmicity suggest a causal connection between these signals and electromechanical alterations in iron-overload-induced cardiomyopathy.