Abstract A cell line, designated FPG, derived from the transplantable rat pituitary tumor MtT/F 4 has been established in long-term culture. The established cell line carries markers similar to those of the original source of material. Several clones from this line have been isolated. Saturable estrogen binding proteins of high affinity were characterized both in the cytosol and in the KCl nuclear extract of the MtT/F 4 tumor growing in castrated animals. Clones C 1FPG and C 5FPG also showed the presence of estrogen receptors in their cytosol. “Empty” resident nuclear receptors in both cloned cell lines were also characterized. As far as can be determined, these nuclear receptors were not translocated from the cytoplasm in an estrogen, temperature-dependent step. The dissociation constants for the estrogen receptor were essentially the same (10 −10 M) in the cytoplasmic and nuclear compartments of both the tumor and the cell lines derived from it. The number of binding sites/cell in the two clones did not vary significantly; they have about 7000–10000 sites/cell. The availability of cell cultures capable of reproducing systems present in whole animals should contribute to an understanding of the mechanism of action of estrogens.