De novo biosynthesis of phosphatidylglycerol and cardiolipin in the isolated intact rat heart was shown to occur from newly synthesized phosphatidic acid via the formation of cytidine-5′-diphosphate-1,2-diacylglycerol (Hatch, G.M. (1994) Biochem. J. 297, 201–208). The biosynthesis of new cardiolipin was investigated in isolated rat hearts perfused with exogenous phosphatidylglycerol. Phosphatidylglycerol was rapidly (within 5 min) incorporated into the heart when hearts were perfused with either phosphatidyl[t4C]glycerol or NBD-phosphatidylglycerol. In hearts perfused with phosphatidyl-[ t4C]glycerol for up to 30 min the amount of radioactivity observed in phosphatidylglycerol was maximum by 5 min of perfusion and remained constant throughout the perfusion period. In the presence of 1–50 μM phosphatidylglycerol, the amount of radioactive phosphatidylglycerol incorporated into the heart was not affected. There was a time-dependent accumulation of radioactivity incorporated into cardiolipin. In addition, radioactivity was incorporated with time into lysophosphatidylglycerol. No significant amount of radioactivity was associated with other phospholipids involved in the biosynthesis of cardiolipin, including phosphatidic acid and cytidine-5′-diphosphate-1,2-diacylglycerol, precursors of cardiolipin biosynthesis via the CDP-DG pathway. We postulate that cardiac cardiolipin may be synthesized from exogenous phosphatidylglycerolindependent of phosphatidylglycerolsynthesized within the heart.