Up-regulation of the high-affinity transport system ( HATS) for NO3- and stimulation of lateral root (LR) growth are two important adaptive responses of the root system to nitrogen limitation. Up-regulation of the NO 3 2 HATS by nitrogen starvation is suppressed in the atnrt2.1-1 mutant of Arabidopsis ( Arabidopsis thaliana), deleted for both NRT2.1 and NRT2.2 nitrate transporter genes. We then used this mutant to determine whether lack of HATS stimulation affected the response of the root system architecture ( RSA) to low NO3- availability. In Wassilewskija (Ws) wild-type plants, transfer from high to low NO3- medium resulted in contrasting responses of RSA, depending on the level of nitrogen limitation. Moderate nitrogen limitation ( transfer from 10 mM to 1 or 0.5 mM NO3-) mostly led to an increase in the number of visible laterals, while severe nitrogen stress ( transfer from 10 mM to 0.1 or 0.05 mM NO3-) promoted mean LR length. The RSA response of the atnrt2.1-1 mutant to low NO3- was markedly different. After transfer from 10 to 0.5 mM NO3-, the stimulated appearance of LRs was abolished in atnrt2.1-1 plants, whereas the increase in mean LR length was much more pronounced than in Ws. These modifications of RSA mimicked those of Ws plants subjected to severe nitrogen stress and could be fully explained by the lowered NO3- uptake measured in the mutant. This suggests that the uptake rate of NO3-, rather than its external concentration, is the key factor triggering the observed changes in RSA. However, the mutation of NRT2.1 was also found to inhibit initiation of LR primordia in plants subjected to nitrogen limitation independently of the rate of NO3- uptake by the whole root system and even of the presence of added NO3- in the external medium. This indicates a direct stimulatory role for NRT2.1 in this particular step of LR development. Thus, it is concluded that NRT2.1 has a key dual function in coordinating root development with external NO3- availability, both indirectly through its role as a major NO3- uptake system that determines the nitrogen uptake-dependent RSA responses, and directly through a specific action on LR initiation under nitrogen-limited conditions.