Nitrate is a major nitrogen source for land plants and also acts as a signaling molecule that induces changes in growth and gene expression. To identify the cis-acting DNA element involved in nitrate-responsive gene expression, we analyzed the promoter of the Arabidopsis gene encoding nitrite reductase (NIR1). A region from positions −188 to −1, relative to the translation start site, was found to contain at least one cis-element necessary for the nitrate-dependent activation of the promoter, in which the activity of nitrate transporter NRT2.1 and/or NRT2.2 plays a critical role. To define this nitrate-responsive cis-element (NRE), we compared the sequences of several nitrite reductase gene promoters from various higher plants and identified a conserved sequence motif as the putative NRE. A synthetic promoter in which the four copies of a 43-bp sequence containing the motif were fused to the 35S minimal promoter was found to direct nitrate-responsive transcription. Furthermore, mutations within this conserved motif in the native NIR1 promoter markedly reduced the nitrate-responsive activity of the promoter, indicating that the 43-bp sequence is an NRE that is both necessary and sufficient for nitrate-responsive transcription. We also show that both the native NIR1 promoter and the synthetic promoter display a similar level of sensitivity to nitrate, but respond differentially to exogenously supplied glutamine, indicating independent modulation of NIR1 expression by NRE-mediated nitrate induction and feedback repression mediated by other cis-element(s). These findings thus define the presence of multiple cis-elements involved in the nitrogen response in Arabidopsis.