The multiple functions of glutamate include regulation of neural development and stem cells. While the importance of the ionotropic glutamate receptors is well established, less is known about the role of metabotropic glutamate receptors (mGluRs). In this study, we examined the effects of pharmacological activation and inhibition of mGluR2/3 on proliferation, differentiation and viability of a human neural stem cell line. Immunofluorescence staining revealed the presence of mGluR2/3 receptors on both proliferating and differentiating stem cells, including cells differentiated into β-tubulin III-positive immature neurons and glial fibrillary acidic protein-positive astrocytes. Stimulation of mGluR2/3 receptors during cell propagation using the agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl) glycine (DCG-IV) increased total cell numbers significantly (60% compared to untreated controls). This effect could be inhibited by the specific antagonist (2S)-2-Amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495). The antagonist alone had no effect. No significant decrease in cell death was found following mGluR2/3 stimulation, suggesting that the observed elevation in cell number was not related to cell viability. Subsequent differentiation of the cells resulted in a slight decrease in beta-tubulin III-positive neurons (5.2% to 3.2% of total cells) for DCG-IV pre-treated cultures. Treatment with DCG-IV and LY342495 during cell differentiation alone had no such effect. Western blot analysis revealed that the active, dimeric form of mGluR2/3 was mainly present on the proliferating cells, which may explain our findings. The present study emphasises the importance of glutamate and mGluRs on regulation of human neural stem cells and suggests a significant role of mGluR2/3 during cell proliferation. This article is protected by copyright. All rights reserved.