Release of glutamine from astrocytes is an essential step of the glutamate-glutamine cycle, and hence for the maintenance of neuronal glutamate and γ-aminobutyric acid (GABA) pools. The glutamine transporter SNAT3 (SN1) has recently been identified as one of the major mediators of glutamine efflux from astrocytes. We investigated the regulation of SNAT3 mediated glutamine transport in cultured astrocytes. Incubation of primary astrocyte cultures with physiological concentrations of glutamate resulted in a rapid, about twofold, upregulation of SNAT3-mediated transport activity. The effect was not mediated by glutamate receptors but required uptake of glutamate into astrocytes. Both net uptake and net efflux increased after treatment of cells with glutamate, excluding an acceleration of the transport by way of an exchange mechanism. Elevated intracellular glutamate most likely reduces the Km of SNAT3 for its substrate glutamine. The results suggest that astrocytes respond actively to the release of glutamate by increasing glutamine release and thereby may modulate glutamatergic neurotransmission. © 2004 Wiley-Liss, Inc.