Flavonoids are divided into flavones, flavonols, flavanones, and isoflavones etc. according to their basal structure, and are known to include compounds with physiological and pharmacological effects such as anti-oxidant, anti-tumor, and anti-inflammation activities. The ingestion of flavonoids may induce pharmacokinetic interactions through the co-administration of drugs. In this study, we investigated the inhibitory potentials on cytochrome P450 (CYP) 3A activity of 23 flavonoids using human liver microsomes, and tried to identify the molecular features that cause the inhibition of CYP3A. The activity of testosterone 6beta-hydroxylate was evaluated to quantify CYP3A activity. We analyzed Quantification Theory I, in which extreme values of the inhibitory effects of CYP3A activity were tested with flavonoids supplied at a level of 10 microM. The inhibitory effects of flavonoids ranged widely from 1.5 microM to more than 100 microM for the half maximal inhibitory concentration. Because the inhibitory effects were only weakly correlated with the pK(a) value, the inhibitory effects could not be accounted for by the molecular characteristics of the flavonoids. On the other hand, flavones with the basal structure and hydroxylation at positions 7 and 4' showed significantly increased inhibitory effects on CYP3A activity. In addition, the hydroxylation of position 2' and 3', methoxylation of position 4', and the isoflavone basal structure significantly decreased the inhibitory effects on CYP3A activity. In conclusion, the basal structure and the substituents of flavonoids are important in the inhibitory effects of flavonoids on CYP3A activity.