We report the results of biosynthetic studies on the sulfur-containing indole phytoalexins, antimicrobial compounds produced by plants after exposure to microorganisms. Feeding experiments with UV-irradiated sliced turnip root (Brassica campestris ssp. rapa) revealed that spirobrassinin (3) is formed via brassinin (1a) and that 1a is biosynthesized by the intramolecular rearrangement of L-tryptophan to form unstable indol-3-ylmethylisothiocyanate (10a). Trapping experiments of 10a with NaSCH3 and formation of PhCH(2)NH-CS-SCH3 (14) on administration of benzyl isothiocyanate supported the presence of the labile isothiocyanate intermediate. Feeding experiments of a mixture of L-[methyl-H-3(3)]methionine and L-[S-35]methionine revealed that the methylthio group of methionine is incorporated into 1a as an unusual intact unit. Incorporation of L-[S-35]cysteine into la and 3 indicated the origin of another sulfur atom. These studies suggest that the biosynthetic pathway of cruciferous phytoalexins is at least partially linked to one of the indole glucosinolates which are typical components of crucifers. Structures of metabolites of 2-methylbrassinin (16) imply the presence of an oxidized intermediate between 1a and other cruciferous phytoalexins.