The antiestrogen tamoxifen is used in the treatment of hormone-responsive breast cancer. However, therapeutic failure has frequently been observed in both patients and animal models after long term treatment. We have studied the effect of a point mutation that leads to the substitution of Val for Gly at codon 400 in the ligand-binding domain of the estrogen receptor (ER) on estrogenic and antiestrogenic activities of 4-hydroxytamoxifen (4-OHT) and its derivatives. Stable ER transfectants derived from MDA-MB-231 CL10A, an ER-negative breast cancer cell line, have been used in these studies. 4-OHT and its fixed ring derivatives showed more estrogen-like activity in ER transfectants than in MCF-7, an ER-positive breast cancer cell line. In this study, 4-OHT was a partial agonist of cell growth in the transfectant S30 cells, which express the wild-type ER. However, it was a full agonist in the mutant ER transfectant ML alpha 2H, which expressed ER with Val at codon 400. The increased estrogenic activity of 4-OHT in ML alpha 2H cells was not due to the preferential isomerization of trans 4-OHT to cis 4-OHT, since the nonisomerizable fixed ring trans 4-OHT was a partial agonist for cell growth in S30 cells and was a full agonist in ML alpha 2H cells. Transient transfection using a reporter plasmid containing an estrogen response element demonstrated that fixed ring trans 4-OHT had estrogenic activity in ML alpha 2H cells.(ABSTRACT TRUNCATED AT 250 WORDS)