Spatial and temporal fluctuations in nitrate (NO3-) availability are very common in agricultural soils. Therefore, understanding the molecular and physiological mechanisms involved in regulating NO3- uptake in regions along the primary root is important for improving the NO3- uptake efficiency (NUpE) in crops. Different regions of maize primary root, named R1, R2 and R3, NO3- starved for 3 days, were exposed to 50 μM NO3-. Electrophysiological measurements (membrane potential and H+ and NO3- fluxes) and NPF6.3, NRT2.1, NAR2.1, MHA1, MHA3 and MHA4 gene expression analyses were carried out. The results confirmed variable spatial and temporal patterns in both NO3- and H+ fluxes and gene expression along the primary maize root. A significant correlation (P = 0.0023) between nitrate influx and gene transcript levels was observed only when NAR2.1 and NRT2.1 co-expression were considered together, showing for the first time the NRT2.1/NAR2.1 functional interaction in nitrate uptake along the root axis.Taken together these results suggest differing roles among the primary root regions, in which the apical part seem to be involved to sensing and signaling in contrast with the basal root which appears to be implicate in nitrate acquisition.