The literature reports that 7α-hydroxydehydroepiandrosterone (7α-OH-DHEA), a metabolite of dehydroepiandrosterone (DHEA), displays numerous anti-aging properties (such as immunostimulation and anti-apoptotic effects) which may result from an apparent antiglucocorticoid activity. However, the molecular mechanism(s) of this effect remain to be elucidated. In the present work, we attempted to unravel some aspects of this mechanism in vitro, in adrenalectomized rats. No specific binding of [3H]-7α-OH-DHEA occurred with the hepatic cytosolic fraction, and the binding of [3H]-dexamethasone ([3H]-DEX) to the cytosolic glucocorticoid receptor complex (GCRC) was unaffected by increasing concentrations of either DHEA or 7α-OH-DHEA. In marked contrast, in isolated hepatic nuclei, the retention of partially purified [3H]-DEX-labelled cytosolic GCRC was significantly decreased after nuclei preincubation with 7α-OH-DHEA, DHEA or 7α-hydroxypregnenolone. However, further experiments using isolated cytosolic fraction preactivated with [3H]-DEX and then filtered on DNA-cellulose columns in the presence or in the absence of 7α-hydroxy steroids unequivocally demonstrated that 7α-OH-DHEA neither competed with the activation of the GCRC, nor inhibited the binding of this complex to DNA-cellulose in the cell-free system. The effect of 7α-OH-DHEA on membrane fluidity of brain cell membranes was observed only at concentrations higher that that of the parent substance DHEA. Thus, the effect of 7α-OH-DHEA does not seem to be mediated by the influence of the accessibility of the hormone to intracellular receptors. While the GCRC binding to DNA is apparently unaffected by 7α-OH-DHEA, and cannot therefore explain the lesser retention of DEX-activated GCRC in isolated nuclei, other mechanisms, possibly extranuclear, such as modification of the conformation of GCRC may be involved. The GCRC in the presence of 7α-OH-DHEA, may account for the antiglucocorticoid properties of this steroid which are currently under investigation.