Abstract: The yeast vacuole is the storage depot for cellular iron. In this paper we quantify the import-export balance in the vacuole due to import of Fe by Ccc1p and to export by the combined activity of Smf3p and the ferroxidase, permease pair of proteins, Fet5p and Fth1p. Our data indicate that the two efflux pathways are equally efficient in trafficking iron out of the vacuole. A major focus of this work was to identify the ferrireductase(s) that supplies the Fe(II) for efflux whether by Smf3p or the Fet5p, Fth1p complex. Using a combination of flameless atomic absorption spectrophotometry to quantify vacuolar and whole cell Fe content and a reporter assay for cytoplasmic Fe we demonstrate that Fre6p supplies Fe(II) to both efflux systems while Fre7p plays no role in Fe-efflux from the vacuole. Enzymatic assay shows the two fusions to have similar reductase activity, however. Confocal fluorescence microscopy demonstrates that Fre6:GFP localizes to the vacuolar membrane; in contrast, Fre7:GFP fusions exhibit a variable and diffuse cellular distribution. Demonstrating a role for the vacuolar metalloreductase Fre6p in Fe-efflux supports the model that iron is stored in the vacuole in the ferric iron state.