The biosynthesis of 5-hydroxy-1,4-naphthoquinone (juglone) was studied by supplying radioactive precursors to leaves of Juglans regia plants. A chemical degradation of the juglone molecule was devised (Fig. 1). With these methods it was shown that the ring atoms of shikimic acid are incorporated into the benzene ring of the quinone, while the carboxyl group of this acid is transformed to 50% into each of the keto groups of the quinone ring (C-atoms 1 and 4 of juglone. Tab. 3). This suggested a symmetrical molecule to be an intermediate in the formation of juglone — most probable 1,4-naphthoquinone. This compound was synthetized with 14C in the positions 2, 3, 9, and 10 and was found to be a good precursor of juglone in Juglans as well as for 2-hydroxy-1,4-naphthoquinone in Impatiens plants (Tab. 4) . 3,4-Dihydroxybenzaldehyde (Tab. 2) and chorismic acid (Tab. 7) which have been suggested previously as intermediates in the biosynthesis of naphthoquinones are no precursors of juglone. The source of three carbon atoms of the quinone nucleus remains to be determined; one or two of these carbon atoms (C2 and/or C3 of juglone) are formed from the methylen carbon of malonate (Tab. 5 and 6); surprisingly, however, the carboxyl carbons of malonic acid are not incorporated. The substitution of shikimic acid occures in the position 6 of this acid as could be judged from the degradation of juglone labelled with shikimic acid [ 1,2-14C] (Fig. 2; Tab. 3). 1,4-Naphthoquinone (or naphthohydroquinone) is postulated as an important intermediate in the biosynthesis of naphthoquinone derivatives in higher plants.