Metabolism and transport from intestinal cells back into the lumen by ATP-binding cassette (ABC) transporters is believed to limit the bioavailability of flavonoids. We studied metabolism and transport of the citrus flavonoid hesperetin, the aglycone of hesperidin, using a two-compartment transwell Caco-2 cell monolayer system, simulating the intestinal barrier. The role of apically located ABC transporters P-glycoprotein (MDR1/ABCB1), multidrug resistance protein 2 (ABCC2), and breast cancer resistance protein (BCRP/ ABCG2) in the efflux of hesperetin and its metabolites was studied by coadministration of compounds known to inhibit several classes of ABC transporters, including cyclosporin A, GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2- isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide], Ko143 [3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a- octahydropyrazino[ 1′,2′:1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tertbutyl ester], MK571 (3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2- dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid), and PSC-833 (Valspodar). Apically applied hesperetin (10 μM) was metabolized into hesperetin 7-O-glucuronide and hesperetin 7-Osulfate, identified using high-performance liquid chromatographydiode array detector (DAD), ultraperformance liquid chromatography-DAD-tandem mass spectrometry, and authentic standards, which were transported predominantly to the apical side of the Caco-2 cell monolayer (1.12 cm2), at average (S.D.) rates of 14.3 (3.7) and 2.1 (0.8) pmol/min/monolayer, respectively. Hesperetin aglycone also permeated to the basolateral side, and this process was unaffected by the inhibitors used, possibly implying a passive diffusion process. Inhibition studies, however, showed that efflux of hesperetin conjugates to the apical side involved active transport, which from the pattern of inhibition appeared to involve mainly BCRP. Upon inhibition by the BCRP inhibitor Ko143 (5 μM), the apical efflux of hesperetin conjugates was 1.9-fold reduced (p ≤ 0.01), and transport to the basolateral side was 3.1-fold increased (p ≤ 0.001). These findings elucidate a novel pathway of hesperetin metabolism and transport and show that BCRP-mediated transport could be a limiting step for hesperetin bioavailability.