In several multidrug resistant tumor cell lines without overexpression of P-glycoprotein (non-Pgp MDR), a decreased accumulation of drugs has been shown to contribute to resistance. We have recently reported that daunorubicin (DNR) accumulation was decreased in the multidrug resistance-associated protein overexpressing GLC4 non-Pgp MDR small cell lung cancer cell line due to an enhanced energy-dependent efflux which could be inhibited by the isoflavonoid genistein. The purpose of this work was 2-fold: (i) to investigate the mechanism by which genistein inhibits the DNR efflux in the GLC4 cells; and (ii) to characterize the dependence of DNR transport on ATP concentration in intact GLC4 cells. The active transport of DNR in GLC4 cells appeared to be a saturable process with an apparent K(m) of DNR of 1.4 ± 0.4 iM. Genistein increased the apparent K(m) value of DNR, suggesting that this agent is a competitive inhibitor of DNR transport. These data provide additional evidence that energy-dependent DNR transport in GLC4 cells is a protein-mediated process. In addition, genistein decreased cellular ATP concentration in a dose-dependent manner in sensitive as well as in resistant cells. Marked inhibition of DNR transport activity in intact GLC4 cells was found when cellular ATP concentration was decreased below 2 mM by sodium azide or 2-deoxy-D-glucose. Thus, since DNR transport in intact GLC4 is already inhibited at modest cellular ATP depletion, a limitation in ATP supply might open ways to make MDR cells more susceptible to drug toxicity.