A chemical genetic approach has been used to investigate the mechanism by which external glutamate (L-Glu) is able to trigger major changes in root architecture in Arabidopsis thaliana L. An initial screen of 80 agonists and antagonists of mammalian glutamate and GABA receptors using a specially developed 96-well micro-phenotyping system found none that either replicated the root’s response to L-Glu or antagonised it. However, a larger screen using >1500 molecules bioactive in yeast identified two groups that interfered with the L-Glu response. One of the antagonists, 2-(4-chloro-3-methylphenyl)-2-oxoethyl thiocyanate (CMOT), has been reported to target Ste11, an evolutionarily conserved MAP kinase kinase kinase (MAP3K) in yeast. This led to the discovery that root growth in a triple mekk1/mekk2/mekk3 mutant (mekk1/2/3), defective in a set of three tandemly arranged MAP3Ks, was almost insensitive to L-Glu. However, the sensitivity of mekk1/2/3 roots to inhibition by other amino acids reported to act as agonists of glutamate receptor-like (GLR) channels in Arabidopsis roots (Asn, Cys, Gly and Ser) was unaffected. L-Glu sensitivity of the mekk1/2/3 mutant was restored by transformation with a construct carrying the intact MEKK1 gene. These results demonstrate that MEKK1 plays a key role in transducing the L-Glu signal that elicits large-scale changes in root architecture and provide genetic evidence for the existence in plants of an L-Glu signalling pathway analogous to that found in animals.