The Aspergillus nidulans stcL gene is predicted to encode a cytochrome P-450 monooxygenase and is located within a cluster of other genes that are required for synthesis of sterigmatocystin. Inactivation of stcL resulted in strains that accumulate dihydrosterigmatocystin, a tetrahydrobisfuran containing molecule that is very similar to the unsaturated product of the wild-type pathway, sterigmatocystin. This observation led us to hypothesize that the A. nidulans sterigmatocystin biosynthetic pathway is branched similarly to the aflatoxin pathway in Aspergillus parasiticus and Aspergillus flavus and that StcL is required for the desaturation of the bisfuran moiety in the sterigmatocystin/aflatoxin precursor versicolorin B. This prediction was confirmed by feeding the stcL mutant with the subsequent pathway intermediate, versicolorin A, which resulted in accumulation of both sterigmatocystin and dihydrosterigmatocystin, indicating that StcL functions before versicolorin A synthesis. A. nidulans stcU was shown previously to encode a ketoreductase required to convert versicolorin A to demethylsterigmatocystin and an stcL, stcU double mutant strain was shown here to accumulate only versicolorin B. These results indicate that both versicolorin A and versicolorin B can serve as substrates for StcU, resulting in a branched pathway. The final product of each branch are sterigmatocystin and dihydrosterigmatocystin, respectively.