N-Trifluoroacetyladriamycin-14-O-hemiadipate (AD 143), a DNA nonbinding derivative of Adriamycin, was studied for its effect on the uptake of labeled nucleosides into human leukemia (ML-1) and lymphoma (P3HR-1) cells in culture. After preincubation with AD 143 at concentrations as low as 5.2 microM (ML-1) or 13 microM (P3HR-1), the ability of the cells to take up extracellular labeled nucleosides was decreased by more than 50%. Similar experiments with Escherichia coli cells showed that AD 143 at the same concentrations did not have any effect. Influx of [3H]thymidine or [3H]uridine was studied by centrifugation of the cells through phthalate oil mixture, and it was found that the influx of the labeled nucleosides was decreased after treatment of the cells with AD 143. An increase in the membrane fluidity was observed after treatment of the cells with AD 143, as revealed by electron paramagnetic resonance spectroscopy studies. These observations suggest that the decreased incorporation of [3H]thymidine and [3H]uridine into acid-precipitable material that we observed earlier in the AD 143-treated cells may in part be the result of the AD 143-induced alteration of cell membrane activities, which in turn causes an inhibition of nucleoside uptake.