Manganese (Mn) toxicity is a major problem for plant growth, often caused by acidic soils leading to an excessive plant availability of Mn2+. However, Mn deficiency is also a major nutritional disorder, commonly observed on slightly alkaline soils, which are favoring oxidation of soluble Mn2+ to the plant unavailable form MnO2. The work reported here was initiated with the aim of identifying and characterizing barley Mn2+ transport proteins. Using yeast as a model system we have identified and characterized barley genes encoding Mn2+ transport proteins. The plasma membrane localized HvIRT1 protein is a broad range metal ion transporter. The HvIRT1 transcript expression was induced under Mn and Fe deficiency and the expression level was correlated with the Mn2+ uptake capacity, suggesting an important role of HvIRT1 in Mn2+ acquisition. Two additional genes encoding Mn2+ transport proteins were identified in a Mn2+ tolerance yeast assay; one belonging to the CDF family designated HvMTP8.1 and one homologous gene to the yeast CCC1, named HvVIT1. It is proposed that HvMTP8.1 has a role in Mn2+ efflux mechanisms into the apoplast whereas HvVIT1 are involved in Mn2+ sequestration into internal cellular compartments. The results presented provide the basis for an improved understanding of the mechanisms involved in Mn2+ acquisition and homeostasis in crop plants. This information will facilitate engineering of genotypes able to grow efficiently on marginal soils.