A high-performance liquid chromatographic method for the assay of diacyl and plasmalogen (alk-1-enyl) phospholipid content and the determination of their fatty acid content from tissue homogenates is described. Myocardial phospholipids are rich in plasmalogens and have a high content of unsaturated fatty acids, including arachidonic acid, esteriffed in the (8 n-2) position. Using a three-stage HPLC assay we have analyzed the phospholipid subclass content and the amount of arachidonic acid esterified to these fractions extracted from isolated perfused rat hearts. After HPLC separation of total myocardial phospholipids, the phosphatidylcholine and phosphatidylethanolamine peak fractions are treated with phospholipase (C) to remove polar head groups and ultraviolet-absorbing benzoate derivatives are made. Separation and quantification of diacyi and plasmalogen content of the total phospholipids with nanomolar sensitivity is then achieved using isocratic elution with a silicic acid HPLC column. The separated plasmalogen and diacyl glycerobenzoates are then subjected to alkaline hydrolysis to remove fatty acids from the (8 n-2) position. The 2-(2,3-napthalimino)ethyltrifluoromethanesulfonate esters of the free fatty acids are then prepared and analyzed with subnanomolar sensitivity using reverse-phase chromatography with gradient elution. As plasmalogen-specific phospholipase (A_{2}) is activated during myocardial ischemia and comprises the majority of total phospholipase (A_{2}) activity in the heart, this methodology allows for a sensitive and complete determination of the changes in the mass of these phospholipids and their arachidonic acid content. (6) 1993 Academic Press, Inc.