Free nuclear receptors were measured in anterior pituitaries from ovariectomized-adrenalectomized rats after extraction of purified nuclei with 0.4 M KC1 and incubation of the extract with 2.5 nM (3H)-estradiol (E2) during 1.5 h at 0-4 degrees C. High affinity, low capacity binding was present in untreated rats; receptor concentration doubled after E2 was given for 4 days, whereas acute (60 min) treatment was without effect. Prolonged exposure to diethylstilbestrol (3 months) down-regulated free as well as total nuclear receptors. Tamoxifen increased free nuclear receptors, induced the progestin receptor in cytosol, and inhibited E2-stimulated serum prolactin but not E2 stimulation of free nuclear receptors. Progesterone and testosterone had no effect on basal or E2-stimulated free nuclear sites, whereas dexamethasone reduced the former but not the latter. Furthermore, from 33 to 36% of free nuclear receptors bound to (3H)-E2, were retained in DNA-cellulose columns whether incubation with ligand was performed at low (0-4 degrees C) or high temperature (25 degrees C); DNA-cellulose binding was unchanged after short E2 but increased after 4 days of E2. These data suggest ligand as well as antiestrogen and corticoid regulation of free nuclear receptors for E2 in anterior pituitary. Free nuclear sites may be a fraction of total cell receptors which are in the transformed state and which bind more tightly to nuclei in vivo, as indicated by the results of DNA-cellulose binding in vitro.