Glucocorticoids can rapidly affect neuronal function and behaviour in mammals. Several studies have suggested the possible existence of rapid, non-genomic effects of glucocorticoids in the hippocampus. To investigate whether glucocorticoids could affect neurotransmission in the hippocampus through rapid, non-genomic mechanisms, we studied the effects of acute glucocorticoid administration on extracellular amino acid levels in the CA1 area of the hippocampus. By means of microdialysis on freely moving rats, we observed that an intraperitoneal injection of corticosterone (2.5 mg/kg) induced a rapid (within 15 min) and transient (returning to basal levels by 35–45 min) increase in extracellular aspartate and glutamate levels (∼ 155–160%), both in sham-operated and adrenalectomized rats. These effects occurred in parallel with a rise in corticosterone concentration, also detected by microdialysis, in this hippocampal area. Intrahippocampal perfusion of corticosterone by retrodialysis also produced the same fast and reversible effects on excitatory amino acid (EAA) levels. Extracellular concentrations of taurine and γ-aminobutyric acid (GABA) were unchanged after intrahippocampal glucocorticoid administration. This corticosterone-mediated rise in EAA levels was not inhibited by the presence of specific antagonists for the two types of intracellular corticosteroid receptors, nor by a protein synthesis inhibitor, anisomycin. Perfusion of dexamethasone, a synthetic glucocorticoid, elicited a similar effect to that observed with corticosterone treatment in all studied cases. However, non-glucocorticoid steroids did not affect amino acid transmission in this hippocampal area. These results indicate that glucocorticoids induce a rapid and transient increase in hippocampal EAA levels in vivo that might be exerted through a novel non-genomic mechanism of action.