Siderophores are low molecular weight compounds, synthesized and secreted by microorganisms, that specifically bind ferric iron with exceptionally high affinity. Microbes capture these compounds and take up the bound iron through specific, high-affinity systems. Saccharomyces cerevisiae can take up iron bound to siderophores through the transporters of the ARN family; however, the mechanism by which the siderophore-bound iron enters the cell via these transporters is not known. Here we describe how ferrichrome, a siderophore of the hydroxamate class, is taken up by Arn1p. Arn1p exhibits two surface binding sites for ferrichrome, one that is similar in affinity to the K(T) for uptake and one of a much higher affinity that is specific for the metallated form of ferrichrome. Ferrichrome may gain access to the higher-affinity site through endocytosis. Tracer studies using (14)C-labeled ferrichrome bound to either iron(III) or aluminum(III), a nonreducible ligand for ferrichrome, indicate that ferrichrome enters the cell as the intact metallosiderophore and accumulates in the cytosol. Both ferrichrome chelates were relatively stable within the cell, and metal-free ferrichrome did not accumulate, indicating a role for ferrichrome in intracellular iron storage. Iron stored as ferrichrome was readily mobilized to meet the metabolic needs of the cell.