The major skeletal rearrangements (anthraquinone - xanthone -coumarin) that occur in the complex biosynthesis of aflatoxin B-1 are mediated by cytochromes P-450. Previous experiments have suggested that two successive monooxygenase reactions are required to convert the xanthone O-methylsterigmatocystin (OMST) to aflatoxin, a process we demonstrate is mediated by a single P-450, OrdA, in Aspergillus parasiticus in accord with findings in A. flavus, The first oxidative cycle is proposed to result in the formation of 11-hydroxy O-methylsterigmatocystin (HOMST), while the second entails aryl ring cleavage, demethylation, dehydration, decarboxylation, and rearrangement to give aflatoxin - a remarkable sequence of transformations. To test this hypothesis, HOMST has been synthesized by an alkylnitrilium variant of the Houben-Hoesch reaction. The troublesome xanthone carbonyl was protected as a butylene to allow further elaboration of the molecule, and then the product xanthone was restored in a uniquely facile peracid deprotection. Methods were devised to construct the sensitive dihydrobisfuran and to maintain the oxidation state of the partially methylated hydroquinone. Expression of ordA in a yeast membrane preparation enabled the intermediacy of HOMST both to be detected in the conversion of OMST to aflatoxin and to be established directly in the biosynthesis of the mycotoxin. Having secured the role of HOMST in aflatoxin formation, the mechanism of the second oxidative cycle of this P-450 is considered.