We previously isolated agosterol A (AG
-A) from a marine Spongia sp. and found that it completely reversed colchicine resistance in P-glycoprotein (Pgp)-over-expressing KB-C2 cells and vincristine resistance in multidrug-resistance protein (MRP)1-over-expressing CV60 cells. However, a tri-deacetylated derivative of AG-A (IAG-A) showed almost no activity in reversing Pgp-or MRP1-mediated drug resistance. In this study, we examined the mechanisms by which AG-A reverses MRP1mediated drug resistance by investigating the interaction between agosterols and MRP1 in MRP1-over-expressing human KB carcinoma (KB/MRP) cells. [ 3 H]-Leukotriene C 4 (LTC 4 ), [ 3 H]-2,4-dinitrophenyl-S-glutathione uptake into membrane vesicles prepared from KB/MRP cells and intracellular [ 3 H]vincristine accumulation and efflux in KB/MRP cells were measured with or without AG-A and/or inactive IAG-A. AG-A reduced MRP1-mediated [ 3 H]-LTC 4 transport in a dosedependent manner, but IAG-A did not. Inhibition by AG-A was competitive, with a K i value of 31 M. AG-A at 10 M enhanced the accumulation of [ 3 H]-vincristine in KB/MRP cells to the level of that in control cells in the absence of the agent. Likewise, ATP-dependent efflux of [ 3 H]-vincristine from KB/ MRP cells was enhanced compared with KB-3-1 cells and inhibited by AG-A. In addition, AG-A reduced intracellular levels of glutathione, a compound required for MRP1-mediated transport of some anti-cancer drugs. These findings suggest that AG-A reverses MRP1-mediated drug resistance by directly inhibiting the capacity of MRP1 to transport drugs. In addition, the capacity of AG-A to reduce cellular glutathione levels may contribute to the modulating activity of MRP1.