Characteristic for cruciferous plants is their production of N-and S-containing indole phytoalexins with disease resistance and cancer-preventive properties, previously proposed to be synthesized from indole independently of tryptophan. We show that camalexin, the indole phytoalexin of Arabidopsis thaliana, is synthesized from tryptophan via indole-3-acetaldoxime (IAOx) in a reaction catalyzed by CYP79B2 and CYP79B3. Cyp79B2͞cyp79B3 double knockout mutant is devoid of camalexin, as it is also devoid of indole glucosinolates [Zhao, Y., Hull, A. K., Gupta, N. R., Goss, K. A., Alonso, J., Ecker, J. R., Normanly, J., Chory, J. & Celenza, J. L. (2002) Genes Dev. 16, 3100 -3112], and isotope-labeled IAOx is incorporated into camalexin. These results demonstrate that only CYP79B2 and CYP79B3 contribute significantly to the IAOx pool from which camalexin and indole glucosinolates are synthesized. Furthermore, production of camalexin in the sur1 mutant devoid of glucosinolates excludes the possibility that camalexin is derived from indole glucosinolates. CYP79B2 plays an important role in camalexin biosynthesis in that the transcript level of CYP79B2, but not CYP79B3, is increased upon induction of camalexin by silver nitrate as evidenced by microarray analysis and promoter--glucuronidase data. The structural similarity between cruciferous indole phytoalexins suggests that these compounds are biogenetically related and synthesized from tryptophan via IAOx by CYP79B homologues. The data show that IAOx is a key branching point between several secondary metabolic pathways as well as primary metabolism, where IAOx has been shown to play a critical role in IAA homeostasis.C haracteristic for cruciferous plants is the synthesis of a wide range of species-specific phytoalexins that structurally are sulfur-containing indole alkaloids (1) and the synthesis of glucosinolates (reviewed in ref.2). Both groups of natural products are involved in plant defense and have cancer-preventive properties (3, 4). Very little is known about the biosynthetic pathway of the S-containing indole phytoalexins. Their similar structure with N-and S-containing side chains at C-3 of the indole ring suggests a biogenetic relationship (5). Camalexin (3-thiazol-2Ј-yl-indole) is produced in the model plant Arabidopsis thaliana (6). It is induced by a variety of microorganisms, e.g., Pseudomonas syringae (6) and Alternaria brassisicola (7), and by abiotic factors, such as AgNO 3 (8). These findings make camalexin a good model compound for studying biosynthesis and regulation of cruciferous indole phytoalexins. In vivo feeding experiments where radiolabeled anthranilate and tryptophan were applied on leaves treated with AgNO 3 led to the suggestion that tryptophan was not a precursor in camalexin biosynthesis because tryptophan was much less efficiently incorporated into camalexin compared with anthranilate (8, 9). The data were further supported by labeling studies performed in three tryptophan mutants (8), where reduced levels of camalexin accumulated in ...