Summary An efficient nitrate uptake system contributes to the improvement of crop nitrogen use efficiency under low nitrogen availability. The H igh A ffinity nitrate T ransport S ystem (HATS) in plants is active in low external nitrate and is mediated by a two-component system [high affinity transporters NRT2 associated to a partner protein NRT3 (NAR2)]. In Brachypodium, the model plant for C3 cereals, we investigated the role of BdNRT2A and BdNRT3.2 through various experimental approaches including gene expression profiling, functional characterisation in heterologous system, intracellular localization by imaging, and reverse genetics via gene silencing. Expression of BdNRT2.A and BdNRT3.2 genes in response to nitrate availability fits with the characteristics of the HATS components. Co-expression of BdNRT2A and BdNRT3.2 is required for an effective nitrate transport in the heterologous expression system Xenopus oocytes. Functional interaction between BdNRT2A-GFP and BdNRT3.2-RFP fusion proteins has been observed at the plasma membrane in Arabidopsis protoplasts in transient expression experiments. BdNRT3.2 appeared to be necessary for the plasma membrane localization of BdNRT2A. 15 Nitrate influx measurements with bdnrt2a mutants (two amiRNA mutants and one NaN 3 induced mutant with a truncated NRT2A protein), confirmed that BdNRT2A is a major contributor of the HATS in Brachypodium. Directed mutagenesis in BdNRT2A of a conserved Ser residue (S461) specific to monocotyledons has been performed to mimic a non-phosphorylated S461A or a constitutively phosphorylated S461D, in order to evaluate its potential role in the BdNRT2A and BdNRT3.2 interaction leading to plasma membrane targeting. Interestingly, the phosphorylation status of S461 did not modify the interaction, suggesting on a more complex mechanism. In conclusion, our data show that BdNRT2A and BdNRT3.2 are the main components of the nitrate HATS activity in Brachypodium (Bd21-3) and allow an optimal growth in low N conditions.