Sterigmatocystin (ST) and aflatoxin B(1) (AFB(1)) are two polyketide-derived Aspergillus mycotoxins synthesized by functionally identical sets of enzymes. ST, the compound produced by Aspergillus nidulans, is a late intermediate in the AFB(1) pathway of A. parasiticus and A. flavus. Previous biochemical studies predicted that five oxygenase steps are required for the formation of ST. A 60-kb ST gene cluster in A. nidulans contains five genes, stcB, stcF, stcL, stcS, and stcW, encoding putative monooxygenase activities. Prior research showed that stcL and stcS mutants accumulated versicolorins B and A, respectively. We now show that strains disrupted at stcF, encoding a P-450 monooxygenase similar to A. parasiticus avnA, accumulate averantin. Disruption of either StcB (a putative P-450 monooxygenase) or StcW (a putative flavin-requiring monooxygenase) led to the accumulation of averufin as determined by radiolabeled feeding and extraction studies.