Human endothelial cells in culture were examined in different growth conditions. The human endothelial cell line, EA.hy 926 cell line, was used and cells were studied either in exponential growth phase, at confluence, or growth-arrested by serum deprivation. Phospholipids were separated and analyzed by high-performance thin-layer chromatography, and their fatty acids were quantified by gas-liquid chromatography. No significant differences in the phospholipid distributions were found between exponentially growing and confluent endothelial cells in which phosphatidylcholine (PC) represented the major phospholipid. In comparison, serum-deprived cells exhibited higher proportions of sphingomyelin and lower content of PC. We also found that among the total lipids, cholesterol level for dividing endothelial cells was lower than for cells growth-arrested either by serum deprivation or by contact inhibition at confluence. The global fatty acid distribution was not affected by the growth conditions. Thus, oleate (18:1 n-9 and 18:1 n-7), palmitate (C16:0), and stearate (C18:0) were the main components of endothelial cell membranes. However, the fatty acid distributions obtained from each phospholipid species differed with the growth status. Altogether, the data indicated that subtle modulations of endothelial cell metabolism appear upon cell growth. The resulting membrane-dependent cellular functions such as cholesterol transport and receptor activities can be expected to be relevant for lipid trafficking within the vessel wall in vitro and in vivo.