Genetic studies have demonstrated that high affinity ferrous transport in Saccharomyces cerevisiae requires an oxidase (Fet3p) and a permease (Ftr1p). Using an iron-independent galactose-based expression system, we show that expression of these two genes can mediate high affinity ferrous iron transport, indicating that these two genes are not only necessary, but sufficient for high affinity iron transport. Schizosaccharomyces pombe also employ an oxidase-permease system for high affinity iron transport. The S. pombe genes, fio1+ (ferrous iron oxidase) and fip1+ (ferriferous permease), share significant similarity to FET3 and FTR1 from S. cerevisiae. Both fio1+ and fip1+ are transcriptionally regulated by iron need, and disruption of fio1+ results in a loss of high affinity iron transport. Expression of fio1+ alone in an S. cerevisiae fet3 disruption strain does not result in high affinity iron transport. This result indicates that the S. pombe ferroxidase, while functionally homologous to the S. cerevisiae ferroxidase, does not have enough similarity to interact with the S. cerevisiae permease. Simultaneous expression of both S. pombe genes, fio1+ and fip1+, in S. cerevisiae can reconstitute high affinity iron transport. These results demonstrate that the oxidase and permease are all that is required to reconstitute high affinity iron transport and suggest that such transport systems are found in other eukaryotes.