The purposes of this study were to determine the effect of structural change on the intestinal disposition of isoflavones and to elucidate the mechanisms responsible for transport of phase II isoflavone conjugates. Transport and metabolism of six isoflavones (i.e., genistein, daidzein, glycitein, formononetin, biochanin A, and prunetin) were studied in the human intestinal Caco-2 model and mature Caco-2 cell lysate. Glucuronides were the main metabolites in intact Caco-2 cells for all isoflavones except prunetin, which was mainly sulfated. In addition, the 7-hydroxy group was the main site for glucuronidation whereas the 4'-hydroxy group was only one of the possible sites for sulfation. Glucuronidated isoflavones (except biochanin A) were preferably excreted to the basolateral side, whereas sulfated metabolites (except genistein and glycitein) were mainly excreted into the apical side. Polarized excretion of most isoflavone conjugates was inhibited by the multidrug resistance-related protein (MRP) inhibitor leukotriene C(4) (0.1 micro M) and the organic anion transporter (OAT) inhibitor estrone sulfate (10 micro M). When formation and excretion rates of isoflavones were determined simultaneously, the results showed that formation served as the rate-limiting step for all isoflavone conjugates (both glucuronides and sulfates) except for genistein glucuronide, which had comparable excretion and formation rates. In conclusion, the intestinal disposition of isoflavones was structurally dependent, polarized, and mediated by MRP and OAT. Formation generally served as the rate-limiting step in the cellular excretion of conjugated isoflavones in the Caco-2 cell culture model.