The binding of the cytoplasmic estrogen receptor (ER c) from N-nitrosomethylurea (NMU)-induced rat mammary tumors to the nucleus using a cell-free system is described. All tumors studied were estrogen-receptor-positive and most of them were hormone-dependent. Sixty-two percent of all tumors investigated ( n = 134) decreased in size more than 30% 4–5 days after ovariectomy. Brief healing of the cytosol loaded with tritiated estradiol induced activation of the ER c measured by an increase of nuclear binding activity. Temperature-dependent activation was evident in every case. The optimal time and temperature of activation were 15–60 min at 30° C. After denaturation of the ER c by heating for 20 min at 56° C only small parts of free estradiol could be bound to nuclei. Mg 2+ ions and EDTA inhibited the nuclear binding of the receptor. The nuclear binding assay was performed for 1 hr at 0–4° C. After this time the activated ER c was bound nearly maximally to nuclei. Under optimized conditions 50–60% of the ER c could be bound to nuclei maximally. Using the same medium for the preparation of crude and purified nuclei the binding of the receptor to both kinds of nuclei was similar. Na 2MoO 4 prevented the activation of the ER c from NMU-tumors completely but did not influence the binding of the previously activated receptor to nuclei.