AbstractThe polyphagous corn earworm Helicoverpa zea frequently encounters aflatoxins, mycotoxins produced by the pathogens Aspergillus flavus and A. parasiticus, which infect many of this herbivore's host plants. While aflatoxin B1 metabolism by midgut enzymes isolated from fifth instars feeding on control diets was not detected, this compound was metabolized by midgut enzymes isolated from larvae consuming diets supplemented with xanthotoxin, coumarin, or indole‐3‐carbinol, phytochemicals that are likely to co‐occur with aflatoxin in infected host plants. Of the two metabolites generated, the main derivative identified in midguts induced with these chemicals and in reactions containing heterologously expressed CYP321A1 was aflatoxin P1 (AFP1), an O‐demethylated product of AFB1. RT‐PCR gel blots indicated that the magnitude of CYP321A1 transcript induction by these chemicals is associated with the magnitude of increase in the metabolic activities of induced midgut enzymes (coumarin>xanthotoxin>indole 3‐carbinol). These results indicate that induction of P450s, such as CYP321A1, plays an important role in reducing AFB1 toxicity to H. zea. Docking of AFB1 in the molecular models of CYP321A1 and CYP6B8 highlights differences in their proximal catalytic site volumes that allow only CYP321A1 to generate the AFP1 metabolite. Arch. Insect Biochem. Physiol. 2008. © 2008 Wiley‐Liss, Inc.