The dependence of uterine progesterone receptor activation on sulfhydryl groups was studied using binding to DNA-cellulose as a measure of activated receptor. Dithiothreitol, thioglycerol, or other sulfhydryl reducing reagents stimulated receptor activation 2- to 3-fold. This effect was produced under mild conditions, i.e. after a 22-h incubation at 0-3 degrees C with 50 mM dithiothreitol. Other characteristics of the sulfhydryl-dependent stimulation of receptor activation, such as pH-dependence and sensitivity to the sulfhydryl blocking agent iodoacetamide, suggest that the sulfhydryl groups essential for receptor activation are different from those involved in steroid binding. Progesterone receptor activation by sulfhydryl reduction was a reversible process, dependent on addition or removal (by dialysis) of the reducing agent. Optimal receptor activation (up to 75% of total receptor) occurred when cytosol was diluted with buffer in the presence of dithiothreitol, suggesting that dissociation of either a receptor subunit or an inhibitory factor present in cytosol may also be involved in the activation process. This putative inhibitor appears to have a Mr greater than or equal to 30,000 since it is not removed from cytosol by dialysis or gel filtration. These results emphasize the importance of sulfhydryl groups or a disulfide bridge, perhaps associated with the DNA-binding domain of the receptor, in a key regulatory step in the mechanism of steroid hormone action: activation and subsequent binding of the steroid-receptor complex to DNA or chromatin.