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An indirect immunofluorescence assay was used to detect estradiol in MCF-7 breast cancer cells to determine if the estradiol-specific fluorescence observed represented estrogen receptor-bound estradiol. Appropriate controls were used to demonstrate the immunological specificity of our assay procedures. Initial studies of estradiol binding in MCF-7 cells were performed at 20 degrees for 1 hr with different concentrations of estradiol. Cytoplasmic and nuclear staining were observed following treatment with 10 nM estradiol, but not with lower concentrations which were nevertheless still sufficient to saturate estrogen receptor. The staining intensity increased with higher estradiol concentration, which is consistent with estradiol binding to lower-affinity binding sites. In order to further determine if estradiol binding by estrogen receptor was being detected, we pretreated MCF-7 cells with 5 nM diethylstilbestrol at 37 degrees for 1 hr to translocate all estrogen receptor to the nucleus and then administered estradiol at varying concentrations for 4 hr at 4 degrees. The estradiol was still primarily detected in the cytoplasm, although virtually all of the estrogen receptor was found to be present in the nucleus by standard [3H]estradiol binding assays. Additional immunochemical studies using sucrose gradient analysis to detect antibody-estradiol-receptor complexes clearly established that these complexes could not be detected. The present results suggest that, although immunocytochemical assays can specifically detect estradiol in MCF-7 cells, the estradiol is bound to lower-affinity binding sites rather than to estrogen receptor. Saturation analyses of intact viable MCF-7 cells performed at 37 degrees for 30 min using [3H]estradiol at concentrations ranging from 0.1 to 93 nM revealed an additional lower-affinity estradiol-binding site besides the receptor, perhaps analogous to the Type II sites reported in the rat uterus and human breast cancers.