Previous reports have suggested that the native hormone-responsive glucocorticoid receptor is a heterocomplex with hsp90 and that the receptor constantly cycles between the hormone-responsive and an inactive state, with complex assembly and turnover being driven by hsp70 and hsp90, respectively. Since hsp70 appears to be titrated in cells that transiently overexpress the receptor, assembly intermediates may accumulate when more receptor is produced than can be assembled to hormone-responsive complex. Comparison of receptor protein and hormone-binding levels in extracts from transiently transfected COS-7 cells revealed the presence of non-hormone-binding receptor forms in addition to the native heterocomplex. The receptor was predominantly nuclear in the majority of the transfected cells even in the absence of hormone, with the DNA-binding domain (DBD) being necessary for nuclear localisation. Moreover, the unliganded receptor exhibited constitutive DNA-binding activity and reactivity towards antibodies against the hinge region where NLS1 is known to reside. By comparing fluorography to immunoblotting of two-dimensional SDS-PAGE of cross-linked [3H]dexamethasone-mesylate-labelled receptor, we detected non-hormone-binding receptor species capable of binding DNA in vitro. In addition, using a constitutively active receptor mutant, we found that the overexpressed wild-type receptor was capable of repressing mutant-activated transcription of transiently and stably transfected reporter genes alike in a DBD-dependent manner.