Iron uptake in Saccharomyces cerevisiae involves at least two steps: reduction of ferric to ferrous ions extracellularly and transport of the reduced ions through the plasma membrane. We have cloned and molecularly characterized FRE2, a gene which is shown to account, together with FRE1, for the total membrane-associated ferric reductase activity of the cell. Although not similar at the nucleotide level, the two genes encode proteins with significantly similar primary structures and very similar hydrophobicity profiles. The FRE1 and FRE2 proteins are functionally related, having comparable properties as ferric reductases. FRE2 expression, like FRE1 expression, is induced by iron deprivation, and at least part of this control takes place at the transcriptional level, since 156 nucleotides upstream of the initiator AUG conferred iron-dependent regulation when fused to a heterologous gene. However, the two gene products have distinct temporal regulation of their activities during cell growth.