Staphylococcus aureus accumulated cardiolipin (CL) and lost phosphatidylglycerol (PG) during the stationary phase of growth. The minor lipids, phosphatidylethanolamine and phosphatidylglucose, also accumulated, whereas the lysylphosphatidylglycerol (LPG) content of the membrane remained constant as stationary phase continued. During exponential growth, the proportions and total content of phospholipids per cell remained constant. The metabolism of the phospholipids was examined under these conditions. In pulse-chase experiments, the phospholipids lost ¹⁴C from the glycerols slower than ³²P. When the phospholipids were labeled with ¹⁴C glycerol, the unacylated glycerols of PG and LPG lost ¹⁴C, whereas the diacylated glycerols either accumulated or did not lose ¹⁴C. In all experiments, the PG showed a more rapid metabolism than the LPG. When staphylococcal CL was hydrolyzed by Haemophilus parainfluenzae CL-specific phospholipase D into phosphatidic acid (PA) and PG, the incorporation of ³²P into both of the phosphates of CL was found to be parallel at both the PG and PA ends of the molecule. However, the specific activity of the ³²P at the PA end was twice that at the PG end of the molecule. The PG end of the CL apparently came from a portion of the cellular PG pool with about 20% the specific activity of the total cellular PG. The turnover of two of the glycerols of the PG portion of CL was like that of the cellular PG. The diacylated glycerol of the PG and of CL and of the membrane PG showed neither turnover nor incorporation of ¹⁴C. Half of the radioactivity was lost from the middle glycerol of CL and the free glycerol of the cellular PG in one bacterial doubling. The diacylated glycerol from the other end of the CL molecule (the PA end) lost radioactivity almost as rapidly as the middle glycerol for 10 min. After the initial rapid loss, the turnover slowed to a rate 10 times slower than the middle glycerol, indicating that the ¹⁴C was actually accumulating at this end of the molecule. The phosphates and glycerols involved in the hydrolysis and resynthesis of the CL molecule during exponential growth in S. aureus apparently come from different pools of PG.