To investigate the regulation of HvNRT2, genes that encode high-affinity NO(3)(-) transporters in barley (Hordeum vulgare) roots, seedlings were treated with 10 mM NO(3)(-) in the presence or absence of amino acids (aspartate, asparagine, glutamate [Glu], and glutamine [Gln]), NH(4)(+), and/or inhibitors of N assimilation. Although all amino acids decreased high-affinity (13)NO(3)(-) influx and HvNRT2 transcript abundance, there was substantial interconversion of administered amino acids, making it impossible to determine which amino acid(s) were responsible for the observed effects. To clarify the role of individual amino acids, plants were separately treated with tungstate, methionine sulfoximine, or azaserine (inhibitors of nitrate reductase, Gln synthetase, and Glu synthase, respectively). Tungstate increased the HvNRT2 transcript by 20% to 30% and decreased NO(3)(-) influx by 50%, indicating that NO(3)(-) itself does not regulate transcript abundance, but may exert post-transcriptional effects. Experiments with methionine sulfoximine suggested that NH(4)(+) may down-regulate HvNRT2 gene expression and high-affinity NO(3)(-) influx by effects operating at the transcriptional and post-transcriptional levels. Azaserine decreased HvNRT2 transcript levels and NO(3)(-) influx by 97% and 95%, respectively, while decreasing Glu and increasing Gln levels. This suggests that Gln (and not Glu) is responsible for down-regulating HvNRT2 expression, although it does not preclude a contributory effect of other amino acids.