Glutamate extracellular levels are regulated by specific transporters. Five subtypes have been identified. The two major ones, GLAST and GLT (glutamate transporters 1 and 2, respectively), are localized in astroglia in normal mature brain. However, in neuron-enriched hippocampal cultures, these proteins are expressed in neurons during the early in vitro development (Plachez et al., 2000). Here, we show that, in these cultures, GLAST and GLT neuronal expression is transient and no longer observed after 7 days in vitro, a stage at which the few astrocytes present in the culture are maturing. Moreover, we demonstrate that these few astrocytes are responsible for the repression of this neuronal expression. Indeed, addition of conditioned medium prepared from primary cultures of hippocampal astrocytes, to cultured hippocampal neurons, rapidly leads to the suppression of neuronal GLAST expression, without affecting neuronal GLT expression. However, when neurons are seeded and co-cultured on a layer of hippocampal astrocytes, they do not develop any immunoreactivity towards GLAST or GLT antibodies. Altogether, these results indicate that glia modulate the expression of GLAST and GLT glutamate transporters in neurons, via at least two distinct mechanisms. Neuronal GLAST expression is likely repressed via the release or the uptake of soluble factors by glia. The repression of neuronal GLT expression probably results from glia-neuron interactions. This further reinforces the fundamental role of direct or indirect neuron-glia interactions in the development of the central nervous system.