A large group of flavonoids was investigated for inhibitory effects on sulfo- and glucurono-conjugation of acetaminophen when added to rat cultured hepatocytes and liver subcellular preparations. The flavonoids inhibited the production of both sulfate and glucuronide conjugates in the cultured cells, with potencies that depended on the specific flavonoid. Among the flavonols, quercetin, kaempferol and galangin were much more effective than myricetin and morin. Flavones including luteolin, apigenin and chrysin were as effective as the corresponding three flavonols above. The inhibition of conjugation by other simple flavones such as 3-, 5-, 7- and 3',4'-OH flavones, and by catechins such as epicatechin and epigallocatechin, was very weak. These data suggest that the presence of both C5 and 7 hydroxyl substitutions on the A-ring in the flavone structure is required for effective inhibitory activity. The effect of flavonoids on sulfo- and glucurono-conjugation was also examined by incubating acetaminophen with isolated liver cytosolic and microsomal preparations, respectively. The active flavonoids in the cells remarkably inhibited the sulfation, but not glucuronidation, in cell-free enzymatic preparations in vitro. The mechanism of inhibition of conjugation by flavonoids in cultured hepatocytes is not likely to depend on the direct inhibition of sulfo- and glucurono-transferase activity by flavonoids.