Iron must cross biological membranes to reach essential intracellular enzymes. Two proteins in the plasma membrane of yeast-a multicopper oxidase, encoded by the FET3 gene, and a permease, encoded by the FTR1 gene-were shown to mediate high-affinity iron uptake. FET3 expression was required for FTR1 protein to be transported to the plasma membrane. FTR1 expression was required for apo-FET3 protein to be loaded with copper and thus acquire oxidase activity. FTR1 protein also played a direct role in iron transport. Mutations in a conserved sequence motif of FTR1 specifically blocked iron transport.