ZIP transporters (ZRT, IRT-like proteins) are involved in the transport of iron (Fe), zinc (Zn) and other divalent metal cations. The expression of IRT3, a ZIP transporter, is higher in the Zn/cadmium (Cd) hyperaccumulator Arabidopsis halleri than is that of its ortholog in Arabidopsis thaliana, which implies a positive association of its expression with Zn accumulation in A. halleri. IRT3 genes from both A. halleri and A. thaliana functionally complemented the Zn uptake mutant Spzrt1 in Schizosaccharomyces pombe; and Zn uptake double mutant zrt1zrt2, Fe-uptake mutant fet3fet4 and conferred Zn and Fe uptake activity in Saccharomyces cerevisiae. By contrast, the manganese (Mn) uptake mutant smf1 phenotypes were not rescued. Insufficient Cd uptake for toxicity was found. Expression of IRT3-green fluorescent protein (GFP) fusion proteins in Arabidopsis root protoplasts indicated localization of both IRT3 proteins in the plasma membrane. Overexpressing AtIRT3 in A. thaliana led to increased accumulation of Zn in the shoot and Fe in the root of transgenic lines. Therefore, IRT3 functions as a Zn and Fe-uptake transporter in Arabidopsis.