Abstract Interaction of [ 3H]4-hydroxytamoxifen-charged estrogen receptor ([ 3H]AER) with nuclei was compared to that of [ 3H]17β-estradiol-charged estrogen receptor ([ 3H]ER) in vitro. Specificity of [ 3H]AER binding was demonstrated since more than 90% of [ 3H]AER binding was displaced by ten-fold excess estradiol-charged ER. For R3230AC tumors, the number of [ 3H]AER binding sites was approximately 40% lower than the number of [ 3H]ER binding sites. There were no differences in affinity of binding of these receptor complexes ( K d range 0.7–1.6 nM). In contrast to a reduction of [ 3H]ER binding after ovariectomy, no difference in the number of [ 3H]AER binding sites was seen among tumors from intact, ovex, or estrogen-treated ovex rats. These results suggest that [ 3H]AER bind to 60% of the sites that bind [ 3H]er, and that neither tissue type nor host ovarian status affects the number of nuclear [ 3H]AER binding sites.