We have studied the effect of membrane structural alteration on the cellular association of the anticancer drug mitoxantrone whose uptake is not carrier-mediated. Membrane fatty acids of L1210 cells were modified by incubating the cells with the highly unsaturated docosahexaenoic acid (22:6), which results in isolated plasma membranes with 37% of the fatty acids as 22:6, or with the monounsaturated oleic acid (18:1), which results in 58% of the fatty acids as 18:1. The rate of uptake by 22:6-enriched cells during the first min was 62% greater than by those enriched with 18:1. The higher rate was recorded at 0.5-16 microM, pH 6.6-7.6 and temperatures 10-40 C. The difference in cell-associated drug apparently was not due simply to a change in mitoxantrone solubility as measured by partitioning of the drug in lipophilic-hydrophilic systems containing lipids from the fatty-acid altered cells. We conclude that the type of fatty acids contained in L1210 cell membranes can affect the cell association of mitoxantrone. This effect could be on transmembrane flux or be due to differences in binding of the drug to intracellular structures.