Estradiol (E), diethylstilbestrol (DES), and three structurally similar DES derivatives and analogs, consisting of racemic mixtures of indenestrol-A (IA; rac), indenestrol-B (IB; rac), and the Z-isomer of pseudo-DES (PD) were used as probes to determine the effect of ligand on receptor-nuclear acceptor site interactions. IA (rac), IB (rac), and Z-PD have been previously shown to interact with the mouse uterine estrogen receptor with high affinity (Kd, approximately 1.5-2.2 x 10(-10) M), occupy similar levels of receptor in the nucleus in vivo, and have nuclear retention times similar to those of E and DES. However, in spite of these similarities, they differentially stimulate estrogenic responses that were previously thought to be interrelated and obligatory for full estrogenic action. In an attempt to elucidate the mechanism of differential stimulation of estrogen-regulated responses, the temporal pattern of KCl-resistant receptor sites was examined after a single injection of E, DES, IA (rac), IB (rac), Z-PD, or saline. Z-PD displayed lower levels of KCl-resistant receptor than the other compounds, whereas IA (rac) and IB (rac) had patterns similar to those of E and DES. In cell-free binding assays, all of the different receptor-ligand complexes were equally effective at competitively inhibiting the binding of receptor-[3H]E complexes to mouse uterine nuclear matrix with a Ki of 1.1 x 10(-10) M. Direct binding analysis showed no difference in the number of nuclear binding sites (range, 40.4-63.9 fmol/100 micrograms DNA) or Kd (range, 1.3-1.8 x 10(-10) M) among the different receptor-ligand complexes. Dissociation studies performed at 4 C showed no differences among the different complexes. In contrast, Z-PD-receptor complexes dissociated much faster from matrix sites at 22 C than any of the other complexes. Significant differences were noted in the proportion of complexes that were resistant to extraction with 0.6 M KCl. Forty-three percent of receptor-E complexes and 44% of receptor-DES complexes were resistant to KCl extraction, while 61% of receptor-IA and 29% of receptor-PD complexes were resistant to extraction, paralleling their activity in eliciting some estrogenic responses. In contrast, no difference was seen by gel shift assays in the ability of the ligand-receptor complexes to bind to a specific PS2 estrogen-responsive DNA sequence. These results demonstrate that the ligand may significantly affect the interaction of the estrogen receptor with nuclear acceptor sites.