Arabidopsis root architecture is highly responsive to changes in the nitrogen supply. External NO3− stimulates lateral root growth via a signalling pathway involving the ANR1 MADS box transcription factor, while the presence of exogenous L-glutamate (Glu) at the primary root tip slows primary root growth and stimulates root branching. We have found that NO3−, in conjunction with Glu, has a hitherto unrecognized role in regulating the growth of primary roots. Nitrate was able to stimulate primary root growth, both directly and by antagonising the inhibitory effect of Glu. Each response depended on direct contact between the primary root tip and the NO3−, and was not elicited by an alternative N source (NH4+). The chl1-5 mutant, which is defective in the NRT1.1 (CHL1) NO3− transporter, was insensitive to NO3− antagonism of Glu signalling, while an anr1 mutant retained its sensitivity. Sensitivity to NO3− was restored in a chl1-5 mutant constitutively expressing NRT1.1. However, expression in chl1-5 of a transport-competent but non-phosphorylatable form of NRT1.1 not only failed to restore NO3− sensitivity but also had a dominant-negative effect on Glu sensitivity. Our results indicate the existence of a NO3− signalling pathway at the primary root tip that can antagonise the root's response to Glu, and they further suggest that NRT1.1 has a direct NO3− sensing role in this pathway. We discuss how the observed signalling interactions between NO3− and Glu could provide a mechanism for modulating root architecture in response to changes in the relative abundance of organic and inorganic N.