The developmentally regulated Aspergillus nidulans yA gene encodes a p-diphenol oxidase that is needed for synthesis of green conidial pigment. We subjected the yA 5' flanking region to mutational analysis in A. nidulans and Saccharomyces cerevisiae to identify DNA sequence elements involved in its transcriptional control, and identified two functionally distinct elements. Element I contained potential BrlA binding sites and was required for full level yA transcription, but not for developmental regulation in the presence of element II. Element II contained putative TEF-1 binding sites flanking a CCAAT element and was sufficient for developmental regulation of transcription. Mutation of the TEF-1 binding sites eliminated developmental regulation, whereas mutation of the CCAAT element led to elevated levels of transcription. Element II was also sufficient to induce transcription in S. cerevisiae when the A.nidulans developmental regulatory gene abaA was expressed from the GAL1 promoter. As AbaA and TEF-1 possess similar DNA binding domains, the abaA-yA interaction in yeast is probably direct. Thus, abaA appears to be a direct activator of yA, but yA regulation may also involve interactions with BrlA and a member of the CCAAT class of DNA binding proteins.