The content and fatty acyl composition of phospholipids were examined in pressure-overload hypertrophied hearts. Cardiac hypertrophy was induced in rats by abdominal aortic constriction. Twenty-one days postconstriction the content of myocardial phosphatidylcholine (PC), sphingomyelin, and phosphatidylinositol (PI) was significantly elevated by 10, 10, and 20%, respectively. The essential fatty acid, linoleic acid, was markedly reduced in PC, phosphatidylethanolamine (PE), PI, and cardiolipin (CL) of hypertrophied hearts. The associated changes in fatty acyl composition were specific for the individual phospholipid class as evidenced by a significant elevation of palmitic acid in PC, docosahexaenoic acid in PE and oleic acid in CL. Alterations in fatty acyl composition of phospholipids were associated with no change in the composition of cardiac triglycerides, cardiac free fatty acids or serum lipids. The fatty acyl composition of phospholipids was also altered in pressure-overload hypertrophied hearts of cats, as evidenced by a reduction of linoleic acid and an elevation of arachidonic acid in total phospholipids. These findings demonstrate that changes in phospholipid metabolism occur in the pressure-overloaded mammalian heart. Such alterations may contribute to altered membrane function in the hypertrophied myocardium.