5-Aminosalicylic acid (5-ASA) is an effective drug for the treatment of ulcerative colitis and Crohn's disease. A large group of flavonoids was investigated for their inhibitory effects on the N-acetyl-conjugation of 5-ASA in rat hepatocytes and subcellular preparations. When added to cultured hepatocytes, some flavonoids inhibited the production of N-acetyl-5-aminosalicylic acid (5-AcASA) with potencies that depended on the specific structure of flavonoids. Among the flavonols, quercetin, kaempferol and galangin had inhibitory activity with a tendency to be more effective at increasing the number of hydroxyl substitutions in the B-ring. Flavones such as luteolin, apigenin and chrysin were as effective as the corresponding three flavonols above. 7,3′,4′-OH flavone was more effective than other simple flavones such as 7-, 5-, 3-, 7,3-, 7,4′- and 3′,4′-OH flavones. Isoflavones were relatively weak inhibitors. Taxifolin and catechins had little or no inhibitory effect. These data suggest that the presence of C7 hydroxyl substitution on the A-ring and the catechol group on the B-ring in the flavone structure is required for effective inhibitory activity. The inhibitory effect of flavonoids on N-acetyl-conjugation of 5-ASA was also examined by incubating 5-ASA with isolated liver cytosolic preparations. The active flavonoids in the cells inhibited the N-acetylation of 5-ASA in the cell-free enzymatic preparations with a potency comparable to that for cultured rat hepatocytes.