Neurotransmitters like glutamate are potential regulators of neurogenesis. Interference with defined glutamate receptor subtypes affects proliferation, migration, and differentiation of neural progenitor cells (NPCs). The cellular targets for the actions of different glutamate receptor ligands are less known. We combined calcium and time-lapse imaging with immunocytochemistry to obtain a spatial overview of the distribution of migrating mouse embryonic neurosphere-derived NPCs responding to glutamate receptor agonists and antagonists and to study the role of these receptors in the motility pattern of NPCs in the neurosphere model. We show that the majority of cells migrating out from neurospheres express metabotropic glutamate receptor 5 (mGluR5) or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors. Responses via mGluR5 correlated with radial glial cells and dominated in the inner migration zones close to the neurosphere. Block of mGluR5 resulted in shorter radial glial processes and a transient increase in neuron-like cells emerging from the neurosphere, and increased the motility of the neuron-like cells. AMPA/kainate receptors are present on the majority of migrating NPCs, which with time accumulate at the outer edge of the migration zone. Blocking these receptors leads to an enhanced extension of radial glial processes and a reduced motility of neuron-like cells. Our results indicate that both mGluR5 and AMPA/kainate receptors are of importance for the guidance of migrating embryonic NPCs and that glutamate, acting on metabotropic and ionotropic receptors, provides essential signals for the motility of NPCs.