The intracellular location of estrogen receptors in hormone-responsive cells has been studied with a number of techniques which indicate that the unoccupied receptors are nuclear and not cytoplasmic proteins. We used cell enucleation of two human breast cancer-derived cell lines, MCF-7 and T47D, to determine whether the unoccupied receptors were also nuclear in these cells and to determine whether the weak estrogen phenol red, present in nearly all tissue culture media, affected the distribution of the receptors seen with this technique. Nucleoplasts prepared from the breast cancer cells contained most of the estrogen receptors that were present in whole cells. The cytoplast fraction, which contained some contaminating whole cells, also contained some receptors. However, incubating cells with estradiol before enucleation did not translocate any receptors out of the cytoplast fraction (to the nucleoplasts). The unoccupied receptors appeared to be almost exclusively nuclear in these cells. The same results were obtained with either radioligand binding or enzyme-linked immunoassay used to measure estrogen receptor, and the distribution of receptors was unaffected by the presence of the pH indicator phenol red. In addition, we observed changes in the estrogen receptor content of incubated cytoplasts that were consistent with receptor synthesis, and this may prove to be a useful model system to characterize receptor synthesis and degradation.