The Aspergillus nidulans aflR gene is found within a 60 kb gene cluster that includes ≈24 other genes that putatively function in the production of the aflatoxin-related mycotoxin sterigmatocystin. Previous work showed that AflR is a C6 zinc binuclear cluster protein that is conserved across Aspergillus spp. and functions as a pathway-specific transcription factor in activating expression of other cluster genes. In this report, we demonstrate that A. nidulans AflR (AnAflR) is a 45 kDa protein that binds to the palindromic sequence 5′-TCG(N5)CGA-3′ found in the promoter regions of several aflatoxin and sterigmatocystin cluster genes (stc genes). The in vivo relevance of this AnAflR binding site was assessed by examining the contribution of the three TCG(N5)CGA elements in the 1.1 kb promoter region of stcU using gene fusions with the bacterial uidA gene encoding β-glucuronidase (GUS). By mutating one, two or all three of the AnAflR-binding elements and examining GUS activity in wild-type aflR or ΔaflR A. nidulans strains, we found that stc gene activation required both AnAflR and at least one TCG(N5)CGA AflR binding site.