Glucocorticoid receptors (GRs) are transcription factors that, upon activation by glucocorticoids, translocate to the cell nucleus, and bind to specific response elements (GREs) in the promoter region of target genes. We analysed stress- and circadian-induced changes in nuclear translocation and GRE binding of GRs in the hippocampus and the prefrontal cortex of the rat brain. Nuclear translocation and binding to GRE were measured in nuclear extracts by Western blot and gel shift, respectively. When glucocorticoid levels were low, as during the light period of the circadian cycle, nuclear GRs and GRE binding were almost undetectable. However, the increase in glucocorticoid levels observed during the dark phase of the circadian cycle or after stress induced a massive nuclear translocation of GRs and GRE binding. These effects were corticosterone-dependent because they were suppressed by adrenalectomy and restored by the injection of corticosterone. Furthermore, GR translocation and GRE binding were of higher amplitude or lasted longer in the hippocampus than in the prefrontal cortex. By contrast, extracellular levels of glucocorticoids, measured by microdialysis in freely moving animals, were identical in the two structures. These results suggest that specific intracellular regulations of GR activity contribute to differentiate the effects of glucocorticoids in different regions of the brain.