The stereochemistry of lignan biosynthesis in Arctium lappa L. is regulated organ-specifically. (+)-Secoisolariciresinol [81% enantiomeric excess (e.e.)] was isolated from A. lappa petioles. In sharp contrast, lignans whose predominant enantiomers have the opposite absolute configuration to that of (+)-secoisolariciresinol [i.e., (-)-matairesinol (>99% e.e.), (-)-arctigenin (>99% e.e.), and (-)-secoisolariciresinol (65% e.e.)] were isolated from seeds of the species. The stereochemical diversity of secoisolariciresinol was demonstrated with enzyme preparations from A. lappa petioles and seeds. Thus, a petiole enzyme preparation catalyzed the formation of (+)-pinoresinol (33% e.e.), (+)-lariciresinol (30% e.e.), and (+)-secoisolariciresinol (20% e.e.) from achiral coniferyl alcohol in the presence of NADPH and H202, whereas that from ripening seeds catalyzed the formation of (-)-pinoresinol (22% e.e.), (-)-lariciresinol (>99% e.e.), and (-)-secoisolariciresinol (38% e.e.) under the same conditions. In addition, the ripening seed enzyme preparation mediated the selective formation of the optically pure (>99% e.e.) (-)-enantiomer of matairesinol from racemic (+/-)-secoisolariciresinols in the presence of NADP. These results indicate that the stereochemical mechanism for lignan biosynthesis in A. lappa varies with organs, suggesting that multiple lignan-synthesizing isozymes are involved in the stereochemical control of lignan formation in A. lappa.