lthough numerous studies have implicated accelerated phospholipid catabolism during myocardial ischemia as an important contributor to ischemic membrane dysfunction, no information is currently available on the subcellular distribution, physical properties, or kinetic characteristics of human myocardial phospholipase A2. In this report, we demonstrate that the overwhelming majority (98%) of total phospholipase A2activity in human myocardium (obtained from transplant recipients) is calcium independent, plasmalogen selective, and is distributed between the microsomal (60–70% of total activity) and cytosolic (30–40% of total activity) fractions. Both human myocardial microsomal and cytosolic phospholipase A2enzymes 1) preferentially hydrolyze plasmalogen molecular species containing arachidonic acid at thesn-2 position, 2) are recalcitrant to chemical inactivation by the indole-reactive agent parabromophenacyl bromide, 3) are irreversibly inhibited by covalent modification of an essential thiol residue by 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), and 4) are exquisitely sensitive to mechanism-based inhibition by (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (bromoenol lactone). In sharp contrast, human mitochondrial phospholipase A21) accounts for only a diminutive amount of total myocardial phospholipase A2activity (1–2%), 2) is augmented by calcium ion, 3) exhibits a higher reaction velocity using phosphatidylcholine in comparison with plasmenylcholine substrate, and 4) is not substantially inhibited by either DTNB or bromoenol lactone. Collectively, these results demonstrate that the majority of phospholipase A2activity in human myocardium is catalyzed by a novel class of calcium-independent plasmalogen-selective phospholipases A2and underscore the potential importance of this class of enzymes in mediating membrane dysfunction during myocardial infarction in humans. (Circulation Research1992;70:486–495)