Salt extracts of purified nuclei from cockerel liver contain a limited number of sites that bind triphenylethylene nonsteroidal antiestrogens with high affinity and specificity. The assay of the [3H]tamoxifen (3H-labeled 1-[4-(2-dimethylaminoethoxy)phenyl] 1,2-diphenylbut-1-(Z)ene)-binding sites is optimally achieved by preincubation of the salt extracts with charcoal-dextran suspension; a 4- to 8-fold increase in activity over that obtained with nontreated extracts is found. This suggests that the binding sites are occupied in vivo by an unknown endogenous ligand. The equilibrium dissociation constant for [3H]tamoxifen binding is 4.76 .+-. 1.8 nM, and the binding site concentration is 1.7 .+-. 0.7 fmol/.mu.g DNA. The concentration of high affinity estrogen-binding sites in the same extracts is almost 30-fold less (0.06 .+-. 0.01 fmol/.mu.g DNA). The relative binding affinities of various antiestrogens for the nuclear antiestrogen-binding sites (with tamoxifen arbitrarily set at 100%) are as follows: nafoxidine (1-[2-(p-[3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl]phenoxy)ethyl]pyrrolidine hydrochloride):126%) > tamoxifen (100%) > N-des-methyltamoxifen (16%) > CI-628 (.alpha.-{p-[2-(1-pyrrolidine)ethoxy]phenyl}4-methoxy-.alpha.''-nitrostilbene; 14%) > 4-hydroxytamoxifen (7%). Estrogens (17.beta.-estradiol, estriol, estrone, and diethylstilbestrol) and several other steroids (cholesterol, dihydrotestosterone, pregnenolone, progesterone and hydrocortisone) show little or no affinity for binding to the nuclear sites (relative binding affinity, < 0.5%). Ether extracts of cockerel serum or liver nuclei contain a substance(s) that competitively inhibits [3H]tamoxifen binding to the nuclear antiestrogen-binding sites. The ether-soluble material does not compete for [3H]estradiol binding to the salt-soluble nuclear estrogen receptor. Cockerel serum and liver nuclei contain a natural compound which competes with the triphenylethylenes at the antiestrogen-binding site and may occupy the nuclear binding sites in vivo.