Nitrate, the major nitrogen source for most plants, is widely used as a fertilizer and as a result has become a predominant freshwater pollutant. Plants need nitrate for growth and store most of it in the central vacuole1. Some members of the chloride channel (CLC) protein family, such as the torpedo-fish ClC-0 and mammalian ClC-1, are anion channels2,3, whereas the bacterial ClC-ec1 and mammalian ClC-4 and ClC-5 have recently been characterized as Cl−/H+exchangers with unknown cellular functions4-6. Plant members of the CLC family are proposed to be anion channels7,8involved in nitrate homeostasis9; however, direct evidence for anion transport mediated by a plant CLC is still lacking. Here we show thatArabidopsis thalianaCLCa (AtCLCa) is localized to an intracellular membrane, the tonoplast of the plant vacuole, which is amenable to electrophysiological studies, and we provide direct evidence for its anion transport ability. We demonstrate that AtCLCa is able to accumulate specifically nitrate in the vacuole and behaves as a NO−3/H+exchanger. For the first time, to our knowledge, the transport activity of a plant CLC is revealed, the antiporter mechanism of a CLC protein is investigated in a native membrane system, and this property is directly connected with its physiological role.