The recurrence every 24 h of glucocorticoid elevation led us to investigate the temporal relationship between glucocorticoid receptor occupation in brain cell nuclei and the availability of cytosol sites. Adrenalectomized rats were injected i.v. with [3H]corticosterone in doses ranging from 15 to 106 nmol/kg. Peak nuclear binding occurred 1-2 h after [3H]corticosterone injection and was preceded by a peak of cytosol receptor labeling at 15-30 min. Yet 1 h after 55 or 105 nmol/kg [3H]corticosterone, no depletion in the total in vitro binding capacity of the cytosol could be detected even though estimated depletion should have been approximately 30% had it occurred. Injection of 80 nmol/kg of corticosterone per rat plus 40 nmol/kg dexamethasone also failed to reduce total cytosol binding capacity, even though estimated depletion should have been approximately 40%. No change in binding affinity of cytosol sites was observed in the injected animals compared to uninjected controls. The in vivo nuclear binding capacity of hippocampus for [3H]corticosterone (fmol/hippocampus) is about 40% of the cytosol binding capacity measured in vitro. Moreover, no more than 40% of total cytosol sites are occupied in vivo as a result of [3H]corticosterone injections which occupy nuclear sites to 80% of estimated capacity. Yet, even with the larger in vitro cytosol binding capacity, a depletion approaching 40% of cytosol binding sites would have been seen, had it occurred as a result of nuclear translocation. The apparent lack of depletion of cytosol receptors is supported by experiments which showed that two injections of [3H]corticosterone 2 h apart fail to fatigue the nuclear uptake mechanism. The present results suggest (1) that in the hippocampus an excess of extranuclear glucocorticoid binding proteins exists, and (2) that the availability of functional cytosol receptors may be regulated to maintain a relatively constant cellular level.