Previous studies have suggested that phospholipid degradation is closely associated with the development of sarcolemmal membrane injury. This study was initiated to characterize the effects of synthetic inhibitors of phospholipase activities using a cultured myocardial cell model in which arachidonic acid is liberated after treatment with the metabolic inhibitor, iodoacetate. Pretreatment with a steroidal diamine (U26,384) blocked the degradation of labeled phosphatidylcholine and the release of arachidonic acid in cultured myocardial cells during ATP depletion. Inhibition of phospholipid degradation by U26,384 prevented the development of sarcolemmal membrane defects and the release of creatine kinase from the cultured myocardial cells during ATP depletion. Pretreatment with U26,384 had no significant effect on the extent of ATP depletion after iodoacetate treatment, which indicates that the activity of this compound could not be simply ascribed to a sparing effect on ATP concentration. These results support the hypothesis that the development of sarcolemmal membrane injury and the associated loss of cell viability are causally related to progressive phospholipid degradation. In addition, these studies indicate that the release of arachidonic acid during ATP depletion is associated with the net loss of the phosphatidylcholine molecule.