Utilizing techniques of Sephadex G-200 column chromatography, sucrose density gradient ultracentrifugation and dextran-coated charcoal adsorption, the characterization of progesterone-binding proteins was attempted in cytosols obtained from the medial basal hypothalamus including the median eminence, the remaining hypothalamus and the cerebral cortex of ovariectomized estrogen-primed rats. After incubation of the medial basal hypothalamic cytosol with 3H-progesterone in the absence or in the presence of various unlabeled steroids, a specific progesterone-binding protein was found in the 7S region with its binding capacity of .apprx. 2 .times. 10-14 mol/mg protein and of .apprx. 6 .times. 10-9 M. No specific binding was found in the remaining hypothalamus and the cerebral cortex.