The ferroxidase Fet3 and the permease Ftr1 constitute a well-conserved high-affinity iron uptake system in yeast. We have investigated the mechanism of transcriptional regulation of Fet3 in the methylotrophic yeast Pichia pastoris. Isolation and functional analysis of the Fet3 promoter indicate that a GATA sequence element plays a role in iron-dependent expression of Fet3. A GATA-type transcription factor, which we have named Fep1, has been partially cloned and it is shown to belong to the family of iron-responsive fungal GATA-factors. These factors share the presence of two Cys(2)-Cys(2) zinc-finger motifs and a set of four conserved cysteines, and are involved in the regulation of siderophore biosynthesis and/or high-affinity iron uptake. Disruption of the FEP1 gene in P. pastoris leads to constitutively high expression of Fet3, irrespective of iron levels, indicating that Fep1 is a transcriptional repressor. EMSA analyses evidence that Fep1 binds to DNA only in the presence of iron.