Abstract In cell-free systems, glucocorticoid receptor-dexamethasone complexes from hepatoma tissue culture (HTC) cells bind to HTC cell nuclei, and estrogen receptor-estradiol complexes from immature uteri bind to uterine nuclei. The binding is specific in that it is of high affinity (apparent Kd = 2 to 3 x 10-10 m) and involves a limited number of acceptor sites (about 3850 per haploid genome). Furthermore, nonradioactive receptor-steroid complexes inhibit the binding of the homologous receptors complexed with tritiated steroid. In each case, the maximum number of complexes bound to isolated nuclei is very similar to the number found in the nuclear fraction of intact cells exposed to saturating concentrations of steroid. Receptor-glucocorticoid complexes also bind with high affinity (apparent Kd = 2.6 x 10-10 m) to about 2150 acceptor sites (per haploid genome) in isolated uterine nuclei, even though the immature uterus is devoid of glucocorticoid receptors. The uterine acceptor sites for glucocorticoid receptors are different from those for estrogen receptors. Thus, the binding of one class of receptor-steroid complex is not inhibited by the other type. The acceptor sites for glucocorticoid receptors in both uterine and HTC cell nuclei are destroyed by DNase, whereas those for estrogen receptors are resistant. Nuclear-bound receptor-dexamethasone complexes are released from both uterine and HTC cell nuclei at a lower concentration of NaCl than is required to release bound receptor-estradiol complexes. Finally, acceptors for estrogen receptors are not found in HTC cell nuclei. It is concluded that nuclear acceptors for glucocorticoid receptors are not restricted to target tissues and therefore nuclei from certain cells are able to bind more than one class of steroid receptor. Where this occurs, however, the results show that each type of nuclear acceptor site binds only one of the two classes of receptor-steroid complex examined. Glucocorticoid and estrogen acceptor sites differ chemically and physically, possibly reflecting differences in the molecular mechanisms of action of these hormones.