The roots of many plant species are known to use inorganic nitrogen, in the form of NO−3, as a cue to initiate localized root proliferation within nutrient-rich patches of soil. We report here that, at micromolar concentrations and in a genotype-dependent manner, exogenous L-glutamate is also able to elicit complex changes in Arabidopsis root development. L-Glutamate is perceived specifically at the primary root tip and inhibits mitotic activity in the root apical meristem, but does not interfere with lateral root initiation or outgrowth. Only some time after emergence do lateral roots acquire L-glutamate sensitivity, indicating that their ability to respond to L-glutamate is developmentally regulated. Comparisons between different Arabidopsis ecotypes revealed a remarkable degree of natural variation in L-glutamate sensitivity, with C24 being the most sensitive. The aux1-7 auxin transport mutant had reduced L-glutamate sensitivity, suggesting a possible interaction between L-glutamate and auxin signaling. Surprisingly, two loss-of-function mutants at the AXR1 locus (axr1-3 and axr1-12) were hypersensitive to L-glutamate. A pharmacological approach, using agonists and antagonists of mammalian ionotropic glutamate receptors, was unable to provide evidence of a role for their plant homologs in sensing exogenous glutamate. We discuss the mechanism of L-glutamate sensing and the possible ecological significance of the observed L-glutamate-elicited changes in root architecture.