We have studied the incorporation of [1-14C]20:5(n-3) and [1-14C]20:4(n-6) in the molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in isolated rat liver cells. These two fatty acids are present in very different amounts in endogenous phospholipids, with 20:4 as one of the major fatty acids and 20:5 as a minor and very diet-dependent constituent. The main phospholipid species formed from 20:4(n-6) were 16:0-20:4 and 18:0-20:4. When formed, they were stable during incubations of liver cells for 2-3 h. The main species formed from 20:5(n-3) were 16:0-20:5 and 18:0-20:5. After formation, 16:0-20:5 and to a lesser degree 18:0-20:5 were, however, degraded during 1-2 h of incubation, especially in PC. Only small amounts of 22:5(n-3) and very little 22:6(n-3) were formed from 20:5(n-3) and small amounts of 22:4(n-6) were produced from 20:4(n-6). With 20:4(n-6) and 20:5(n-3) as substrates, 20:4-20:4 and 20:5-20:5 molecular species respectively were initially formed in PC and PE but both species were rapidly degraded.