Characterization of estrogen-binding components was attempted in cytosol fractions from thymus, spleen and mesenteric lymph node of castrated rats. As shown by sucrose gradient analysis, specific binding of [6, 7-3H]estradio1-17/3 in the thymus is associated with a component migrating at 4 S. The binding of [6, 7-31-flestradio1-17,8 is highly specific since it is easily displaced by unlabeled estradio1-17i5 and diethylstilbesterol.Affinity of unlabeled estrone, estriol and clomiphene citrate, is much lower, and estradiol-17a, progesterone, testosterone, 5a-dihydrotestosterone and corticosterone have no affinity for the component at all. The dissociation constant of thymic estrogen binding is 0.25 nm in males and 0.3 nm in females.The number of binding sites is 6 fmols/mg protein in both sexes. No specific binding to estrogen is, however, found in cytosol fractions from the other two tissues.Enzyme-and heat-experiments demonstrate that specific estrogen binder in thymic cytosol is heat-labile and at least protein in nature.It is concluded that the rat thymus contains a cytoplasmic estrogen receptor which is in part protein and heatlabile.