Human umbilical vein endothelial cells were cultured in various fatty acid-modified media until equilibrium conditions were reached (7-8 days). The effects on the fatty acid composition of phospholipid classes and on the metabolism of arachidonic acid (20:4(n-6)) were studied. The results showed that in every phospholipid class large changes in fatty acid composition, including 20:4(n-6) content, were induced by long-term modification with unsaturated as well as saturated fatty acids. However, the mean levels of saturated and unsaturated fatty acids per phospholipid class remained relatively constant, except for cells modified with oleic acid, which showed an increase in monounsaturated fatty acids at the expense of both saturated and polyunsaturated fatty acids. The rate of incorporation of radiolabeled 20:4(n-6) in endothelial lipids was not influenced by long-term fatty acid modification. Cells modified with 20:4(n-6) (having a high 20:4(n-6) content) tended to "store" excess 20:4(n-6) as the elongated product 22:4(n-6) mainly in phosphatidylserine and ethanolamine phospholipids. On the other hand, endothelial cells modified with 20:5(n-3) (having a low 20:4(n-6) content) differed typically from other fatty acid-modified cells by a relatively high level and high incorporation rate of 20:4(n-6) in phosphatidylinositol, with a low extent of elongation. These results indicate extensive homeostatic control of membrane unsaturation in each phospholipid class and economical control of 20:4(n-6) content in all modified endothelial cells, irrespective of a considerable variation of 20:4(n-6) levels in cellular lipids. Moreover, the observed maintenance of a critical level of 20:4(n-6) in phosphatidylinositol, when 20:4(n-6) supply was strongly decreased, may be important for maintaining proper signal transduction upon endothelial cell stimulation.