The purpose of this study was to determine whether bioflavonoid glucoside O-conjugates are absorbed from the intestine in the intact form or as their aglycones following hydrolysis by intestinal β-glucosidases. In this study, the intestinal absorption of genistin, the β-glucoside of the isoflavone genistein, was examined in anesthetized, adult female rats fitted with indwelling biliary cannulas. To first establish whether genistein, once absorbed, was converted into unique metabolites, genistin was infused into the femoral or portal veins and bile samples quantitatively collected. Analysis of bile samples by HPLC-mass spectrometry revealed that almost full recovery of the genistein component occurred in the form of unreacted genistin (∼20%) and genistein 7β-O-glucuronide (∼80%). However, when genistin was infused into the upper small intestine, only genistein 7β-O-glucuronide and the aglycone genistein appeared in the bile. There was no evidence for any biliary secretion of the unreacted genistin, thereby excluding its uptake in the intact form from the small intestine in this animal model. © 2006 American Society for Nutrition.