Extensive intestinal glucuronidation has been previously reported in both human and animals after oral administration of naturally occurred flavonoids. The present study aims to investigate the relationship between human intestinal glucuronidation activity and the position of hydroxyl substitution on flavonoids. Seven commercially available mono-hydroxyflavones (HF), namely 3-, 5-, 6-, 7-, 2'-, 3'- and 4'-mono-hydroxyflavones, were chosen as model compounds. Glucuronidation activity of the selected seven HFs was investigated by incubating each HF at various concentrations with human jejunum S9 at 37 degrees C for 10 min. The generated glucuronides were identified by HPLC/MS and quantified by HPLC/UV. Metabolic kinetics parameters including Km and Vmax of each HF were determined. The results demonstrated that the glucuronidation activity of 6- and 3'-mono-hydroxyflavones was much greater than that of 3-, 4'-, 7- and 2'-HF with 5-HF to be the lowest. The findings imply that nucleophilicity and stereo-conformation of OH substituents are crucial for the intestinal glucuronidation of flavonoids.