Sulfation and glucuronidation are the principal metabolic pathways of flavonoids,and extensive phase Ⅱ metabolism is the main reason for their poor bioavailabilities.The purpose of this study was to compare the similarities and differences in the positional preference of glucuronidation versus sulfation in the mouse liver S9 fraction.The conjugating rates of seven mono-hydroxyflavones (HFs) (i.e.,2-,3-,4-,3-,5-,6-,and 7-HF),and five di-hydroxyflavones (diHFs),(i.e.,6,7-,4,7-,3,7-,5,7-,and 3,4-diHF) were determined in three separate enzymatic reaction systems:(A) sulfation only,(B) glucuronidation only,or (C) simultaneous sulfation and glucuronidation (i.e.,Sult-Ugt co-reaction).In general,glucuronidation rates were much faster than the sulfation rates.Among the HFs,7-HF was the best substrate for both conjugation reactions,whereas 3-HF was rapidly glucuronidated but was not sulfated.As a result,the rank order of sulfation was very different from that of glucuronidation.Among the diHFs,regiospecific glucuronidation was limited to 7-OH and 3-OH positions,whereas regiospecific sulfation was limited to 7-OH and 4-OH positions.Other positions (i.e.,6-OH and 5-OH) in diHFs were not conjugated.The positional preferences were essentially maintained in a Sult-Ugt co-reaction system,although sulfation was surprisingly enhanced.Lastly,sulfation and glucuronidation displayed different regiospecific-and substrate-dependent characteristics.In conclusion,glucuronidation and sulfation shared the same preference for 7-OH position (of flavonoids) but displayed unique preference in other positions in that glucuronidation preferred 3-OH position whereas sulfation preferred 4-OH position.