Zinc (Zn) is an essential element for plants but limited information is currently available on the molecular basis for Zn(2+) transport in crop species. To expand the knowledge on Zn(2+) transport in barley (Hordeum vulgare L.), a cDNA library prepared from barley roots was expressed in the yeast (Saccharomyces cerevisiae) mutant strain Deltazrt1/Deltazrt2, defective in Zn(2+) uptake. This strategy resulted in isolation and identification of three new Zn(2+) transporters from barley. All of the predicted proteins have a high similarity to the ZIP protein family, and are designated HvZIP3, HvZIP5 and HvZIP8, respectively. Complementation studies in Deltazrt1/Deltazrt2 showed restored growth of the yeast cells transformed with the different HvZIPs, although with different efficiency. Transformation into Fe(2+) and Mn(2+) uptake defective yeast mutants showed that the HvZIPs were unable to restore the growth on Fe(2+) and Mn(2+) limited media, respectively, indicating a specific role in Zn(2+) transport. In intact barley roots, HvZIP8 was constitutively expressed whereas HvZIP3 and HvZIP5 were mainly expressed in -Zn plants. These results suggest that HvZIP3, HvZIP5 and HvZIP8 are Zn(2+) transporters involved in Zn(2+) homeostasis in barley roots. The new transporters may facilitate breeding of barley genotypes with improved Zn efficiency and Zn content.