A cDNA encoding a pinoresinol lariciresinol reductase PLR (PLR-Lp1) from a cell culture of Linum. perenne was cloned. Hairy root lines were transformed with an ihpRNAi construct to suppress PLR-Lp1 gene expression. As a second enzyme of the proposed pathway, Justicidin B 7-hydroxylase (JusB7H) was characterized from a microsomal fraction prepared from a L. perenne H suspension culture. The enzyme catalyzes the last step in the biosynthesis of Diphyllin by introducing a hydroxyl group in position 7 of Jus B. Enantiomeric purity in lignan biosynthesis is determined during the first steps, but on different levels. Seeds of Linum usitatissimum contain almost 99% (+)- and 1% (-)-Seco-diglucoside. A recombinant PLR (PLR-Lu1) from L. usitatissimum seeds converts only (-)-pinoresinol (Pino) to (+)-Seco. A second cDNA PLR-Lu2 from the leaves of L. usitatissimum was isolated. The recombinant protein converts only (+)-Pino to (-)-Seco. Therefore, the enantiomeric composition of lignans in the organs of L. usitatissimum is determined by the relative action of two PLRs with opposite enantiospecificity rather than a single enzyme with low enantiospecificity.