Although lipids are largely involved in cardiovascular physiopathology, the lipid metabolism in endothelial cells remains largely unknown. Human umbilical vein endothelial cells (HUVECs) were used to investigate the metabolism of complex lipids. The membrane phospholipid homeostasis results from both de novo synthesis and remodelling that ensures the fine tuning of the phospholipid fatty acid composition. Using [ 3 H]-glycerol and phosphoderivatives we showed the efficiency of glycerolipid synthesis from glycerol (0.9 nmol h−1 mg proteins−1 ), but not from its phosphorylated form suggesting the requirement of a functional glycerol kinase in HUVECs. Conversely, the synthesis of triacylglycerols was very low (less than 5% of phospholipid synthesis). The incorporation rate of fatty acids into phospholipids showed that there is a specific fate for each fatty acid in respect to its chain length and saturation level. Moreover in steady state condition, increasing the long chain ω3 polyunsaturated fatty acids in the medium resulted in an increased polyunsaturated/saturated ratio in phospholipids (from 0.42 to 0.63). [ 14 C]O 2 was produced form either [ 14 C]-glucose or [ 14 C]-palmitate indicating the functionality of the oxidation pathways, although β-oxidation was less efficient than glucose oxidation. The endothelial cell lipid metabolism involves conventional pathways, with functional rates largely slower than in hepatocytes or in cardiomyocytes.