3H-Dexamethasone binding sites with a Kd of approximately 0.7 nM and a maximum number of binding sites of approximately 0.3 pmoles/mg protein were demonstrated in the uterine cytosol of adrenalectomized rats only if dithiothreitol was present in the incubation mixture and the simultaneous presence of molybdate further enhanced the binding in the cytosol. The binding sites exhibited a high specificity for glucocorticoids and were depleted in a dose-dependent manner from cytosol after administration of dexamethasone to animals. The depletion was not due to the occupation of the binding sites by the dexamethasone administered and the rate of depletion was correlated with the inhibition of uterine growth induced by estrogen administration. The cytosol labeled with 3H-dexamethasone in the presence of dithiothreitol bound to DNA-cellulose efficiently after heating at 25 degrees C for 30 min and the binding was inhibited by pyridoxal 5'-phosphate added to the reaction mixture. The effect of heating on the DNA-cellulose binding was abolished by molybdate in the incubation mixture. From these observations, it was concluded that 3H-dexamethasone binding sites in the rat uterus were physiologically active glucocorticoid receptors.