Diabetes-induced changes in phospholipase A(2) (PLA(2)) activity have been measured in several tissues but are undefined in diabetic myocardium. We measured ventricular PLA(2) activity in control, streptozotocin-induced diabetic, and insulin-treated diabetic rats and characterized myocardial phospholipids to determine whether diabetes altered myocardial phospholipid metabolism. Increased membrane-associated Ca(2+)-independent PLA(2) (iPLA(2)) activity was observed in diabetes that was selective for arachidonylated phospholipids. Increased iPLA(2) activity was accompanied by an increase in choline lysophospholipids. Diabetes was associated with marked alterations in the phospholipid composition of the myocardium, characterized by decreases in esterified arachidonic and docosahexaenoic acids and increases in linoleic acid. The decrease in polyunsaturated fatty acids was confined to diacylphospholipids, whereas the relative amount of these fatty acids in plasmalogens was increased. Diabetes-induced changes in PLA(2) activity, lysophospholipid production, and alterations in phospholipid composition were all reversed by insulin treatment of diabetic animals. Diabetes-induced changes in membrane phospholipid content and phospholipid hydrolysis may contribute to some of the alterations in myocardial function that are observed in diabetic patients.