Glucuronidation and sulfation are the two major phase II metabolic pathways for flavones, natural compounds that hold great potential for improving human health. We investigated the positional preference for sulfation and glucuronidation of seven structurally similar flavones in vitro and in situ. An FVB mouse intestinal perfusion model was used in addition to three small intestine S9 fractions catalyzing sulfation only (Sult enzymes), glucuronidation only (Ugt enzymes) or both (Sult and Ugt enzymes). In both the single and co-reaction S9 systems, flavones containing 7-OH groups were conjugated only at 7-OH despite the presence of other hydroxyl groups, and 7-OH glucuronidation was faster than sulfation (P < 0.05). The sulfation rate was enhanced in the Sult-Ugt co-reaction system, while glucuronidation was usually unchanged by the presence of Sult. In the intestinal perfusate, sulfation patterns were the same in the small intestine and colon, and the excretion rate of 7-O-sulfate was the fastest or second fastest. The excretion of 7-O-glucuronidates was faster in small intestine (P < 0.05) than in colon. The S9-mediated sulfation rates of the different flavones were significantly correlated with the excretion rates of the same flavones from perfused intestine. In conclusion, flavone glucuronidation and sulfation rates were sensitive to minor changes in molecular structure. In intestinal S9 fractions, both Ugts and Sults preferentially catalyzed reactions at 7-OH. The sulfation rate was significantly enhanced by simultaneous glucuronidation, but glucuronidation was unaltered by sulfation. Sulfation rates in mouse S9 fractions correlated with sulfation rates in perfused intestine. Copyright © 2011 John Wiley & Sons, Ltd.