Based on quantitative high-performance liquid chromatographic analyses of molecular species in selected phospholipid subclasses from culture human umbilical vein endothelial cells, the relative degree of unsaturation was ethanolamine plasmalogens greater than phosphatidylethanolamine greater than phosphatidylcholine. A total of 36 different molecular species were identified in the phosphatidylcholine fraction. Interestingly, the phosphatidylcholine contained a significant amount (11.7%) of the dipalmitoyl species, a lipid normally associated with lung surfactant. The arachidonoyl-containing molecular species of phosphatidylserine/inositol were labeled to the highest extent and the ethanolamine plasmalogens contained the lowest specific radioactivity after incubating [3H]arachidonic acid with human endothelial cells for 4 h. Within each phospholipid subclass the arachidonoyl species where both acyl groups of the phospholipid are unsaturated (20:4-20:4, 18:2-20:4 + 16:1-20:4, and 18:1-20:4) had higher specific radioactivities, after labeling with [3H]arachidonic acid, than those that contained saturated aliphatic chains (16:0-20:4 and 18:0-20:4). This indicates that the unsaturated species have higher turnover rates.