Isolated rat hepatocytes were suspended and stored in either Liebovitz- 15 medium (37 °C or 4 °C) or University of Wisconsin (UW) solution (4 °C) containing [3H] arachidonic acid (AA). At varying times, membrane phospholipids were separated by thin layer chromatography. AA labeled phospholipids similarly at both 4 °C and 37 °C. Analysis of the ratios of [3H] AA and [14C] glycerol incorporated into phosphatidic acid or other phospholipids in dual-labeled cells indicated that the deacylation/reacylation cycle was the major route of AA incorporation at hypothermia. This was supported by showing that blocking phospholipase A2 (PLA2) activity by trifluoperazine suppressed AA incorporation into phospholipids. PLA2 activity, measured by determining the release of AA, was slow during 48-hour cold storage, but increased significantly when ATP was depleted by inhibition of mitochondria and glycolysis. In the whole rat liver, there was no significant loss of phospholipids during 48-hour storage (total phospholipids [μmol phosphorus/L/mg]: 0.197 ± .001 at 0 hours) unless energy blockers were used (0.155 ± .005 at 48 hours) or glycogen depleted by fasting the rat (0.167 ± .001 at 48 hours). This study shows that a net PLA2 stimulated hydrolysis of phospholipids is seen only when ATP is depleted and its generation from anaerobic glycolysis inhibited. Thus, PLA2 hydrolysis of phospholipids is not a significant cause of liver cell injury during cold storage when livers are obtained in optimal condition. However, conditions affecting the generation of ATP during cold storage could alter PLA2 leading to membrane damage.