Abstract Nucleo-cytoplasmic distribution of estrogen receptors and DNA polymerase α activity in human endometrial adenocarcinoma cells (HEC-50 line) was evaluated after separation of nuclei following either homogenization or enucleation with cytochalasin B. About 30% of the estrogen receptor was found in the nuclear fraction after homogenization whereas 86% was found in the karyoplasts after enucleation. The total amounts of estrogen receptor per cell after homogenization and enucleation were not significantly different (14,000–17,000 binding sites/cell). Receptor measurements were carried out using the hydroxylapatite method after labeling with [ 3H]estradiol (5nM [ 3H]E 2 ± 500 nM E 2) at 30°C for 3 h. About 20% of the DNA polymerase α activity was found in the nuclear fraction after homogenization, whereas 96% was found in the karyoplasts after enucleation. The average total activity (0.84 Units/10 6 cells) in homogenized cells was about 1 8 of the activity in karyoplasts. These results indicate that estrogen receptor and DNA polymerase α activity reside in the nucleus in intact HEC-50 cells. DNA polymerase α is translocated to the cytoplasmic fraction and inactivated after homogenization.