A pure glucocorticoid agonist RU 28362 and the potent antagonist RU 38486 were compared with dexamethasone for the evolution and the molecular nature of the GR during insulin-dependent conversion of 3T3-F442A preadipocytes into mature cells. In the whole cell assay system, the affinity for preadipocyte GR was observed in the order RU 38486 > RU 28362 > dexamethasone. The GR complex was most stable in presence of dexamethasone follwed by the antagonist RU 38486 = the agonist RU 28362. Similar results were obtained in mature adipocytes but the binding of RU 38486 was more equivocal. An insulin-dependent differentiation process did not alter any of these parameters but increased the number of GR nearly fivefold over a 2-week period. Ion-exchange analysis of the cytosolic receptor revealed that the differentiation process was not accompanied by the appearance of any novel or new forms of GR, contrary to the situation in the liver, since both RU 38486 and dexamethasone were bound to identical molecular species of GR. These data provide a defined system for further analysis of cellular receptor as a function of steroid, tissue, and species, contrary to the classical dogma where GR is generally thought to be identical as a passive vehicle for the steroid in all circumstances, and affinity for steroid is generally equated with receptor stability.