AbstractPrimary cultures of astrocytes free of neurons and containing less than 1% of oligodendrocytes were examined for their ability to incorporate labeled precursors into lipids and glycolipids. At selected developmental stages cultures were doublelabeled with either [3H]glycerol and [14C]acetate or with [3H]galactose and Na2[35SO4] for a total of 72 hr. Lipids were extracted with CHCl3/CH3OH, fractionated on a silicic acid column, and further resolved by two‐dimensional thin‐layer chromatography. It was found that (1) cultured astrocytes actively incorporate acetate and glycerol into various phospholipids; (2) they have very limited ability to utilize galactose and virtually lack the synthetic machinery to use Na2SO4for the synthesis of sulfated sphingogalactolipids; and (3) their overall lipid metabolism is very distinct from that of oligodendrocytes. It was also found that cultured astrocytes have low levels of 2′,3′ cyclic phosphodiesterase and glycerol phosphate dehydrogenase activities; the latter is less than one fifth of that in oligodend