Under limiting growth conditions, Aspergillus nidulans produces a carcinogenic secondary metabolite related to aflatoxin and called sterigmatocystin (ST). The genes for ST biosynthesis are co-ordinately regulated and are all found within an approximately 60-kilobase segment of DNA. One of the genes within this region is predicted to encode a CX2CX6CX6CX2CX6CX2 zinc binuclear cluster DNA-binding protein that is related to the Aspergillus flavus and Aspergillus parasiticus aflatoxin regulatory gene aflR. Deletion of the A. nidulans aflR homolog resulted in an inability to induce expression of genes within the ST gene cluster and a loss of ST production. Because A. nidulans aflR mRNA accumulates specifically under conditions that favor ST production we expect that activation of ST biosynthetic genes is determined by A. nidulans aflR. In support of this hypothesis, we demonstrated that induced expression of the A. flavus aflR gene in A. nidulans, under conditions that normally suppress ST gene expression, resulted in activation of genes in the ST biosynthetic pathway. This result demonstrates that AflR function is conserved between Aspergillus spp. and that aflR expression is sufficient to activate genes in the ST pathway.