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Aflatoxins, the most toxic and carcinogenic family of fungal secondary metabolites, are frequent contaminants of foods intended for human consumption. Previous studies showed that formation of G-group aflatoxins (AFs) from Omethylsterigmatocystin (OMST) by certain Aspergillus species involves oxidation by the cytochrome P450 monooxygenases, OrdA (AflQ) and CypA (AflU). However, some of the steps in the conversion have not yet been fully defined. Extracts of Aspergillus parasiticus disruption mutants of the OYE-FMN binding domain reductase-encoding gene nadA (aflY) contained a 386 Da AFG1 precursor. A compound with this mass was predicted as the product of sequential OrdA and CypA oxidation of OMST. Increased amounts of a 362 Da alcohol, the presumptive product of NadA reduction, accumulate in extracts of fungi with disrupted aryl alcohol dehydrogenase-encoding gene norB. These results show that biosynthesis of AFG1 involves NadA reduction and NorB oxidation.