Matairesinol is a central precursor in planta in the biosynthesis of numerous lignans, including that of the important antiviral and anticancer agent, podophyllotoxin. In this study, the approximately 32-kDa NAD-dependent secoisolariciresinol dehydrogenase, which catalyzes the enantiospecific conversion of (-)-secoisolariciresinol into (-)-matairesinol in Forsythia intermedia, was purified >6,000-fold to apparent homogeneity. The 831-base pair cDNA clone encoding this 277-amino acid protein was next obtained from a library constructed from F. intermedia stem tissue, whose fully functional recombinant protein, produced by expression of this cDNA in Escherichia coli, catalyzed the same enantiospecific conversion via the corresponding lactol intermediate. A homologous secoisolariciresinol dehydrogenase gene was also isolated from a Podophyllum peltatum rhizome cDNA library, whose 834-base pair cDNA clone encoded a 278-amino acid protein with a calculated molecular mass of approximately 32 kDa. Expression of this protein in E. coli produced a fully functional recombinant protein that also catalyzed the enantiospecific conversion of (-)-secoisolariciresinol into (-)-matairesinol via the intermediary lactol. Various kinetic parameters were defined and established conversion of the intermediary lactol as being rate-limiting. With this overall enzymatic conversion now unambiguously defined, the entire biochemical pathway to the lignans, secoisolariciresinol and matairesinol, has been elucidated. Last, both secoisolariciresinol and matairesinol are metabolized in the gut of mammals, following digestion of high fiber dietary grains, seeds, and berries, into the so-called "mammalian" lignans, enterodiol and enterolactone, respectively; these in turn confer significant protection against the onset of breast and prostate cancers.