The intermolecular, regio- and stereoselective phenol coupling for the biosynthesis of the bicoumarin kotanin in Aspergillus niger has been investigated. Feeding experiments with singly and doubly (13)C-labeled monomeric precursors clearly proved that it is not the coumarin siderin but its hydroxy derivative, demethylsiderin, that undergoes phenol coupling. However, siderin is demethylated regioselectively to demethylsiderin and it is the latter that is coupled to the corresponding dehydrodimer, orlandin. The product is subsequently O-methylated in a stepwise fashion to demethylkotanin and kotanin. Crude extracts were analysed by HPLC with chemically synthesized bicoumarins as reference compounds. This and a stereochemical analysis of the isolated bicoumarins revealed that A. niger produces exclusively the (P)-atropisomers of the three 8,8'-bicoumarins, kotanin, demethylkotanin, and orlandin. The absence of other monomeric or dimeric coumarins strongly suggests an intermolecular, regio- and stereoselective mode for the phenol-coupling step.