CYS3 is the positive-acting global regulatory protein involved in the sulfur control circuit in Neurospora crassa and belongs to the family of bZIP DNA-binding proteins. Here we report a characterization of native DNA-binding sites recognized by CYS3. DNA footprinting experiments and systematic mutational analysis were used to define the consensus CYS3-binding sequence, 5'-ATGPuPyPuPyCAT, a 10-bp palindrome. The sequence 5'-ATGACGTCAT acts as a strong binding site, and all single nucleotide changes within this sequence resulted in a reduction, or even complete loss, of CYS3 DNA-binding. Site-directed mutagenesis was employed to study two uncharged residues, serine 113 and phenylalanine 116, in the basic region of the CYS3 protein bZip DNA-binding domain. Ser113 appears to be directly involved in a specific interaction with nucleotide 2 of the binding site, possibly by making a direct contact with this base, and Phe116 contributes significantly to DNA-binding affinity.