The effect of a serum-free medium (TNB-100), compared to RPMI 1640 containing 10% fetal bovine serum (FBS), on the lipid composition of HL60 and K562 leukemic cells was investigated. The 10% FBS RPMI medium contained approximately three times more phospholipids (PL), about three times more protein and eight times more cholesterol (CHOL) than did the TNB-100 medium. Cells cultured in TNB-100 medium, referred to as HL60-TNB and K562-TNB cells, were significantly lower in PL and CHOL than 10% FBS RPMI cells, with about a threefold higher PL-to-CHOL ratio; however, these cells were significantly higher in protein content. Cells grown in TNB-100 were also significantly more fluid than 10% FBS RPMI cells and were more sensitive to the fluidizing action of the ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine. The 50% inhibitory dose of the drug was about 50% lower in TNB-grown cells than in 10% FBS RPMI cells.