Acyl turnover of rat hepatocyte phospholipids and triacylglycerols was assessed by incubating the cells in media containing 40% H2(18)O and measuring the time-dependent incorporation of 18O into ester carbonyls by gas chromatography-mass spectrometry of hydrogenated methyl esters. Incorporation of 18O into 22-carbon acyl groups was low in phosphatidylcholine, phosphatidylinositol, and phosphatidylserine, whereas in phosphatidylethanolamine, it was about the same as in the other acyl groups. Incorporation of 18O into individual molecular species of phosphatidylcholine and phosphatidylethanolamine was determined after phospholipase C hydrolysis, derivatization to dinitrobenzoates, and separation by high-performance liquid chromatography. In most molecular species, acyl groups at the sn-1 and sn-2 positions became 18O-labeled at drastically different rates, indicating remodeling through deacylation-reacylation. Molecular species expected to arise de novo from acylation of glycerophosphate exhibited similar rates of 18O incorporation at the sn-1 and sn-2 positions. The data suggest that hepatocyte phospholipids are continually synthesized, remodeled by deacylation-reacylation at specific turnover rates up to 10-15%/h, and degraded. This acyl turnover probably does not involve the majority of intracellular unesterified fatty acids whose 18O incorporation was found to be very low. In contrast, the oxygens of extracellular unesterified fatty acids were readily exchanged with the media. This exchange was enzyme-catalyzed, possibly by lipases released into the media from damaged cells. Incorporation of 18O into exogenously added fatty acids was also rapid and resulted in enhanced uptake of 18O-labeled fatty acids into cellular lipids, primarily triacylglycerols and phosphatidylcholine, without drastic change of the intracellular free fatty acid pool.