During screening for the flavonoid chemosensitizers, it was found that 5,7,3',4',5'-pentamethoxyflavone (PMF) was equipotent to verapamil in vitro with respect to the chemosensitizing effect. PMF appears to have a chemosensitizing effect not only by increasing the intracellular accumulation of the drugs without competition in a binding site of azidopine but also by interfering with the substrate-stimulated ATPase activity. Structure-activity relationship suggests that methoxylated substitution and its numbers or sites of the rings are more important than its hydroxylated counterparts in chemosensitization. Overall, PMF is anticipated to be a novel and highly potent second-generation flavonoid chemosensitizer because PMF has significant advantages of having a high therapeutic index, of being a non-transportable inhibitor, and of having a low possibility of drug interactions at the azidopine-binding site of Pgp. (C) 2004 Elsevier Inc. All rights reserved.