The epipodophyllotoxin glucopyranosides have previously been shown to interact with membrane lipids and to alter the activity of several lipid-embedded membrane proteins. To determine if these agents are acting as general membrane perturbants, we have further examined their effects on membrane processes in Ehrlich ascites tumor cells. [3H]VM-26 and [3H]VP-16 were taken up rapidly and concentrated within the cells in proportion to their lipophilicity. Neither agent was found to have any significant effect on the influx of L-[3H]leucine or alpha-[3H]aminoisobutyric acid. Likewise, these drugs had no significant effects on the hexose transporter. The nucleoside transporter, which is structurally and functionally similar to the hexose transporter, was dramatically affected, however. VM-26 was a non-competitive inhibitor of equilibrium-exchange influx of cytosine arabinoside in Ehrlich cells with a Ki of 15 microM. Equilibrium-exchange influx increased with temperature in control cells (Q10 = 2) but not in VM-26-treated cells; thus, VM-26 was a more potent inhibitor at higher temperatures. VM-26 also significantly reduced zero-trans influx in Ehrlich, P388, L5178Y, and ML-1 cells, and these effects were immediate in onset. VM-26 inhibited high-affinity binding of the nucleoside transport inhibitor nitrobenzylmercaptopurine riboside (NBMPR), but VM-26 enhanced non-specific NBMPR binding to Ehrlich cells. The apparent specificity of the epipodophyllotoxins for the nucleoside transporter is discussed.