Previous studies on Fe(II) uptake in Saccharomyces cerevisiae suggested the presence of two uptake systems with different affinities for this substrate. We demonstrate that the FET3 gene is required for high affinity uptake but not for the low affinity system. This requirement has enabled a characterization of the low affinity system. Low affinity uptake is time-, temperature-, and concentration-dependent and prefers Fe(II) over Fe(III) as substrate. We have isolated a new gene, FET4, that is required for low affinity uptake, and our results suggest that FET4 encodes an Fe(II) transporter protein. FET4's predicted amino acid sequence contains six potential transmembrane domains. Overexpressing FET4 increased low affinity uptake, whereas disrupting this gene eliminated that activity. In contrast, overexpressing FET4 decreased high affinity activity, while disrupting FET4 increased that activity. Therefore, the high affinity system may be regulated to compensate for alterations in low affinity activity. These analyses, and the analysis of the iron-dependent regulation of the plasma membrane Fe(III) reductase, demonstrate that the low affinity system is a biologically relevant mechanism of iron uptake in yeast. Furthermore, our results indicate that the high and low affinity systems are separate uptake pathways.