Ethyl acetate, n-butanol and water extracts were prepared from a hot-water extract of Paeony root, which shows strong antimutagenic activity for benzo [α] pyrene (B [α] p). Among them, ethyl acetate and n-butanol extracts showed the antimutagenicity. The ethyl acetate extract was fractionated to 20 fractions (Fr.1-20) with Sephadex LH-20, and the n-butanol extract to 6 fractions (Fr.21-26) with preparative high performance liquid chromatography (HPLC). Antimutagenic activity was observed in Fr.2, Fr.9-12, and Fr.22. Gallic acid (GA) was found in Fr.2 and Fr.22, and pentagalloyl glucose (PGG) in Fr.9-12. The amounts of GA and PGG in Paeony root extract were determined by HPLC, and the antimutagenic activity of these extracts were attributable to GA and PGG. Antimutagenic effects of GA and PGG were explored in the following points. They are (I) reaction and adsorption of B [α] p with GA and PGG, (II) inhibition of the enzymatic activity of S9mix by GA and PGG, (III) inactivation of the metabolites of B [α] p by GA and PGG, and (IV) bio-antimutagenic effect of GA and PGG on DNA damaged bacteria. GA and PGG inactivated the metabolites of B [α] p, and PGG inhibited the enzymatic action of S9mix. Their inhibitory effect was recognized as desmutagenic action.