Several homologous genes encoding proteins involved in regulating siderophore synthesis in fungi have been isolated, including the sre gene from the filamentous fungus Neurospora crassa. We present data that further characterize SRE and provide new insights into the regulation of iron homeostasis in Neurospora. SRE is a member of the GATA factor family, which is comprised of transcription factors that contain either one or two zinc finger motifs that recognize and bind to GATA-containing DNA sequences. Results from electrophoretic mobility shift assays demonstrate that SRE binds with high affinity to a DNA probe containing the iron response element from the sid1 promoter from Ustilago. SRE binding to DNA was demonstrated to be zinc-dependent. Moreover, changes in the spacing between two GATA sites altered the DNA binding affinity of SRE. Mutants of highly conserved cysteine residues present in SRE and homologous proteins were created by site-directed mutagenesis. The combined results of mobility shift assays, siderophore synthesis assays, and ornithine oxygenase enzyme activity determinations demonstrate that these mutants with cysteine substitutions have a dominant repressor phenotype.