This paper describes studies on the fatty acid composition of individual phospholipids of the neonatal rat cardiomyocyte as well as in the gas-dissected sarcolemma derived from those cells. There is a sarcolemmal fatty acid asymmetry between the two leaflets of the membrane, which results from an asymmetric phospholipid distribution and particular fatty acid composition of each phospholipid class. The cytoplasmic leaflet is shown to be more unsaturated than the outer one. The phospholipids preferring the inner sarcolemmal leaflet (PE, PS, and PI) are particularly rich in two fatty acids, stearic acid and arachidonic acid. The implications of the data in current models for Ca2+ binding and for disruption of sarcolemma following ischemia and reperfusion damage are discussed.