1. 1. The lipid composition of L-M cells, a variant of L-cells that can be grown in a lipid-free, chemically defined medium in the absence of serum, has been determined. The effect of 10% horse serum, a requirement for growth of regular L-strain fibroblasts, on the lipid composition of the L-M cell has also been investigated. The lipid classes of the L-M cells grown on horse serum are quantitatively similar to those of the L-M cells grown on the chemically defined medium; the lipid composition of the cells grown under both conditions is different from that of the horse serum. However, L-M cells utilize fatty acids from the serum since large levels of linoleic acid are found in the lipid classes of those cells grown on horse serum. 2. 2. The L-M cells and the tumors induced by injecting L-M cells into mice are rich sources of glyceryl ethers. Glyceryl ether diesters are most abundant in the L-M cells grown on the chemically defined medium (20% of the neutral lipids), whereas the tumors contain approx. 8% glyceryl ether diesters in the neutral lipid fraction. The phospholipids of L-M cells grown with and without serum and of L-M tumors contain near equal percentages of glyceryl ethers (12.3-15.9%). The O-alkyl and O-alk-1-enyl moieties are saturated or monounsaturated and have chain lengths of 16 and 18 carbon atoms. © 1969.