Glutamate is the main neurotransmitter at hippocampal mossy fibre (MF) terminals. Because neurotransmitters have been proposed as regulating factors of neural network formation and neurite morphogenesis in the developing CNS, we examined the possible contribution of glutamate to MF pathfinding. Entorhino-hippocampal slices prepared from early postnatal rats were cultivated in the presence of glutamate receptor antagonists. Timm histochemical staining revealed that pharmacological blockade of metabotropic glutamate receptors (mGluR), but not of ionotropic glutamate receptors, induced abnormal outgrowth of the MFs. When slices were cultured in the presence of mGluR antagonists, DiI-labelled MF axons displayed a great degree of defasciculation, and MF-mediated EPSPs in the CA3 pyramidal cells were altered. Similar results were obtained for a selective antagonist of group II mGluR, but not of group I or III mGluR. Glutamate is, therefore, likely to regulate MF outgrowth via activation of group II mGluR. The present study may provide a novel role of glutamate in hippocampal development.