Glucocorticoids (GCs) are known to act via binding to and activating intracellular GC receptors (GR) expressed by target cells. The activated GR then translocates from the cytoplasm into the nucleus to regulate target gene transcription. GCs readily enter the brain and are known to regulate many aspects of neuronal function. Therefore intracellular regulation of GR activity may potentially play an important role in modulating the effects of GCs in this central target tissue. We report that after a single bolus I.V. injection of corticosterone (cort) in the rat, activated GR rapidly translocates from the cytoplasm into the nucleus of cells in the hippocampus (HC) and pre-frontal cortex (PFC). The appearance of GR within the nucleus is rapidly followed by a clearance of GR from the nucleus. This appears to be a proteasome-dependent machanism because pretreatment with the specific irreversible 26S proteasome inhibitor MG132 abolishes the decrease in nuclear GR levels. Male Sprague-Dawley rats (n = 3/timepoint/group) were anaesthetised with I.M. injection of Hypnorm (fentanyl citrate 0.252 mg/kg and fluanisone 8 mg/kg) after I.P. injection of Diazepam (4 mg/kg) then subjected to bilateral adrenalectomy, jugular cannulation, and I.C.V. cannulation into the lateral ventricle. After recovery for 5 days, rats were infused via the I.C.V. cannula with either 1 µl of 100 μM MG132 in 1% DMSO/saline or 1% DMSO/saline as vehicle alone, at 5pm. After 16 h, the rats were given a bolus I.V. injection of 100 µg cort. Animals were killed at times 0, 15 and 120 min. The HC was rapidly dissected and frozen. Nuclear extracts were prepared and analysed by Western blot with the GR M20 antibody (Santa Cruz) to determine nuclear GR levels throughout the timecourse in the presence and absence of the inhibitor. I.V. injection of 100 μg cort resulted in a rapid increase in plasma GC levels at 1 min (883±157 ng/ml), with subsequent rapid decrease consistent with the 10 min half-life of cort in blood. Subsequent nuclear translocation of GR in the HC occurred maximally at 15 min after injection (13.1±3.5-fold). Interestingly, the retention time of GR in the nucleus was short-lived, with levels decreasing at 30 min and reaching near-baseline levels by 60 min. In contrast, in the rats treated with MG132 there was little decrease in levels of nuclear GR in the HC, with levels staying high at 120 min after I.V. bolus injection of cort. Densitometry analysis revealed levels of GR detected in the nucleus to be 9.0 ± 2.6-fold higher than baseline (time 0) in the presence of MG132, compared to 0.9 ± 0.1-fold in the absence of MG132 (significant difference in unpaired t-test, p=0.0199). Our results suggest that the 26S proteasome is involved in rapidly degrading activated GR after it translocates into the nuclei of GR-expressing cells in the HC. The functional significance of proteasome-dependent rapid turnover of activated GR in the nucleus may be to allow dynamic and continuous responses to the highly fluctuating hormone levels observed in pulsatile GC secretion.