Irinotecan (CPT-11) is a widely-used potent anticancer drug that inhibits mammalian DNA topoisomerase I, however overexpression of the ATP-binding cassette transporter ABCG2 can confer cancer cells resistance to SN-38, the active form of CPT-11. In the present study, we have examined the contribution of three variant forms of ABCG2 to SN-38 resistance. Exogenous expression of the Arg482 (wild type), Gly482, and Thr482 variants of ABCG2 conferred HEK293 cells resistance to SN-38 by 15.0-, 5.0-, and 5.3-fold, respectively. In plasma membrane vesicles prepared from the ABCG2 variant cDNA-transfected HEK293 cells, [Arg482]ABCG2 transported SN-38 and its glucuronide conjugate in an ATP-dependent manner; however, only minimal transport activities were observed with the other variants (Gly482 and Thr482). In addition, we have screened natural flavonoids to find potent inhibitors of [Arg482]ABCG2. Quercetin was found to be the strongest inhibitor (Ki = 0.28 microM) among natural flavonoids tested in the plasma membrane system in this study. When [Arg482]ABCG2-transfected HEK293 cells were incubated with SN-38 in the presence of 20 microM quercetin, cellular resistance to SN-38 was partly reversed. In this context, certain flavonoid derivatives are considered to be good candidates for development of ABCG2 inhibitors.