We have investigated the fatty acid composition of brain capillary endothelial cells cultured alone or in coculture with astrocytes, using an in vitro model in which endothelial cells and astrocytes were grown from one part of a filter to another. We found that the fatty acid composition of the cocultured cerebral endothelial cells was markedly different from that of non-cocultivated endothelial cells. The most striking difference was the increase of arachidonic acid (20:4n-6) at the expense of its precursor, linoleic acid (18:2n-6). Similar modifications were found for the n-3 family of fatty acids with an increase of docosahexaenoic acid (22:6n-3) at the expense of its precursors, but the differences were less than within the n-6 fatty acids. These changes induced by the coculture were observed only in endothelial cell phospholipids, especially the phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine classes, but were not detected in phosphatidylinositols and in other lipid classes. Only the composition of the n-3 series fatty acids was altered in another capillary endothelial cell type (from adrenal cortex) cocultured with astrocytes under the same conditions. The fatty acid changes observed might be biologically relevant as they tended to make the fatty acid composition of the brain capillary endothelial cells more closely resemble that of brain microvessels.