Lipid synthesis and its regulation by serum lipoproteins at the microvascular blood-brain barrier were studied using primary cultures of microvascular endothelial cells from rat brain. These cells are capable of synthesizing all their lipids (neutral lipids, phospholipids, glycolipids) from the water-soluble compounds, glucose, acetate, acetoacetate and beta-hydroxybutyrate. The ketone bodies, especially acetoacetate, are the preferred substrates for lipid synthesis. The incorporation patterns of acetate, acetoacetate and beta-hydroxybutyrate are very similar, indicating that these precursors contribute to lipid synthesis via the same metabolic route. However, the metabolic pathway is different for glucose, which is preferentially incorporated into phospholipids. The existence of an inverse relationship between lipid synthesis and the serum lipoprotein concentration suggests that cultured cerebral endothelial cells are capable of taking up lipids, principally cholesterol, contained in the serum lipoproteins. Cellular lipids would thus be supplied both by intracellular lipid synthesis and by serum lipoproteins. The difference between cholesterol synthesis rates in cultured cerebral endothelial cells and in isolated brain microvessel cells could be partly explained by the fact that the lipoprotein concentration is much lower in the culture medium than in rat serum.