Lateral root initiation is strongly repressed in Arabidopsis by the combination of high external sucrose and low external nitrate. A previously isolated mutant, lin1, can overcome this repression. Here, we show that lin1 carries a missense mutation in the NRT2.1 gene. Several allelic mutants, including one in which the NRT2.1 gene is completely deleted, show similar phenotypes to lin1 and fail to complement lin1. NRT2.1 encodes a putative high-affinity nitrate transporter that functions at low external nitrate concentrations. Direct measurement of nitrate uptake and nitrate content in the lin1 mutant seedlings established that both are indeed reduced. Because repression of lateral root initiation in WT plants can be relieved by increased concentrations of external nitrate, it is surprising to find that repression is also relieved by a defect in a component of the high-affinity nitrate uptake system. Furthermore, lateral root initiation is increased in lin1 relative to WT even when seedlings are grown on nitrate-free media, suggesting that the mutant phenotype is nitrate-independent. These results indicate that NRT2.1 is a repressor of lateral root initiation and that this role is independent of nitrate uptake. We propose that Arabidopsis NRT2.1 acts either as a nitrate sensor or signal transducer to coordinate the development of the root system with nutritional cues.