HvALMT1 from barley (Hordeum vulgare L.) encodes a protein capable of facilitating the transport of malate and other organic anions when expressed in Xenopus oocytes. The HvALMT1 gene is primarily expressed in guard cells of stomata, in regions behind the root apex and at lateral root junctions. We investigated the function of HvALMT1 in planta by overexpressing it in barley under the control of a constitutive promoter. Transgenic plants expressing HvALMT1 at levels four to 9-fold greater than controls showed reduced growth and plants showing the highest expression failed to set seed. Although measurements of conductance indicated that stomatal function was not totally impaired in the transgenic plants the time taken for the stomata to close in response to low light was significantly longer compared with controls. Elemental and metabolomic analyses of the transgenic barley shoots revealed that the concentration of calcium and levels of ascorbate, serine, threonine and pentanoate were consistently greater (2- to 14-fold) in plants that overexpressed HvALMT1, whereas whole-shoot tissue levels of fumarate were significantly lower (60-85% reduction). Transgenic plants also showed significantly greater efflux of malate and succinate from their roots than control plants. Efflux of these organic anions occurred independently of Al3+ and conferred greater Al3+ resistance in solution culture and in acidic soil. These results are consistent with HvALMT1 contributing to anion homeostasis in the cytosol and osmotic adjustment by transporting organic anions out of the cell or by sequestering them into cytosolic vesicles.