Ether-linked glycerophospholipids (ether lipids, EL) are selectively toxic and anti-proliferative agents against cancer cells in vitro. The reason for such selectivity is not completely clear. Their mechanism of action is mediated through an interaction with the plasma membrane and the membrane lipid composition may modulate it. As a continuation of previous reports, we now present data showing that cholesterol concentration modulates EL toxicity in the K562, U937 and MOLT4 leukemic cell lines in vitro. Cells become sensitive to otherwise ineffective doses of EL when their cholesterol content is lowered. Cell cholesterol levels were reduced by exposure to an egg lipid mixture (neutral glycerides, phosphatidylcholine and phosphatidylethanolamine, AL721). The data contribute to an understanding of the EL mechanism of action on membranes and suggest that the cellular cholesterol concentration must be considered a major factor in modulating the cytotoxic effects of EL.