Using an optimized precursor feeding system, the biosynthesis of the alkaloid lunarine in Lunaria annua seeds was investigated. The synthesis of radioactively labelled precursors for the application experiments is described. Lunarine was shown to be synthesized by stereo-selective phenol-oxidative coupling of N-1,N-10-bis(p-coumaroyl)spermidine. p-Coumaric acid for the biosynthesis of this dimer is formed from L-phenylalanine via trans-cinnamic acid. The polyamine moiety of lunarine, spermidine, and its immediate precursor putrescine are preferentially synthesized from arginine. Enzyme studies with microsomes of Lunaria annua seeds indicated that a cytochrome P-450 enzyme might be responsible for the phenol-oxidative coupling of N-1,N-10-bis(p-coumaroyl)spermidine yielding the hexahydrodibenzofuran ring of lunarine. Copyright (C) 1997 Elsevier Science Ltd. All rights reserved.