Aspergillus flavus isolates produce only aflatoxins B1 and B2, while Aspergillus parasiticus and Aspergillus nomius produce aflatoxins B1, B2, G1, and G2. Sequence comparison of the aflatoxin biosynthesis pathway gene cluster upstream from the polyketide synthase gene, pksA, revealed that A. flavus isolates are missing portions of genes (cypA and norB) predicted to encode, respectively, a cytochrome P450 monooxygenase and an aryl alcohol dehydrogenase. Insertional disruption of cypA in A. parasiticus yielded transformants that lack the ability to produce G aflatoxins but not B aflatoxins. The enzyme encoded by cypA has highest amino acid identity to Gibberella zeae Tri4 (38%), a P450 monooxygenase previously shown to be involved in trichodiene epoxidation. The substrate for CypA may be an intermediate formed by oxidative cleavage of the A ring of O-methylsterigmatocystin by OrdA, the P450 monooxygenase required for formation of aflatoxins B1 and B2.