Multidrug resistance due to P-glycoprotein is a serious impediment to successful chemotherapy of cancer. Numerous compounds are known that inhibit the drug-exporting function of P-glycoprotein. Understanding the mechanisms of action of these chemosensitizers is made difficult by the complexity of the in vivo cell systems usually employed. To examine the direct effects of chemosensitizers, we have developed a system in which purified and reconstituted P-glycoprotein transports. Hoechst 33342 from the lipid membrane to the aqueous interior of proteoliposomes, requiring ATP hydrolysis (Shapiro AB and Ling V, J Biol Chem 270: 16167-16175, 1995). Here, we use this system to understand the effect on P-glycoprotein of quercetin, one of three flavonoids that have been reported to have the unique property of stimulating drug transport by P-glycoprotein in vivo (Phang et al., Cancer Res 53: 5977-5981, 1993). Since flavonoids are abundant in food, it is important to understand their effects on the function of P-glycoprotein because of the implications for cancer chemotherapy. In our hands, quercetin inhibited P-glycoprotein-mediated Hoechst 33342 efflux and enhanced accumulation, as measured by flow cytometry, by multidrug-resistant CHRC5 cells. In the purified system, quercetin strongly inhibited Hoechst 33342 transport by P-glycoprotein, at least in part by inhibiting the ATPase activity of P-glycoprotein required for transport. We conclude that the previously reported stimulatory effect of quercetin on drug efflux from multidrug-resistant cells is not a direct effect on P-glycoprotein. The ATPase domain of P-glycoprotein may be an attractive target for new chemosensitizing agents.