Abstract: 【Abstract】 Objective To detect protein dynamic changes of cellular localization and the DNA damage response of epithelial ovarian cancer cells to chemo-radiotherapy. Methods 28 specimens of epithelial ovarian cancer were collected, with 6 cases diagnosed as borderline serous cystadenoma, 5 as highly differentiated, 6 as medium differentiated and 11 as poorly differentiated cystadenocarcinoma. Collagenase A was used for digesting tissues before primary culture. We compared the characteristics of cells cultured in different mediums (MCDB/M199 medium, primary culture medium, and DMEM medium) supplemented with multiple growth-promoting factors. The characteristics of cells were examined in terms of the maintenance of normal cell morphology, proliferation potential, and cell fibrosis proteins （53BP1 and γ-H2AX） responsive to DNA damage 〔those in the ATM checkpoint pathway determined by indirect immunofluorescent staining after treatment with camptothecin (CPT) and X-ray〕. Results Normal morphology was maintained relatively well in the cells cultured in MCDB/M199 medium and its cell fibrosis was slow compared with the cells cultured in other media. Gradually increased endogenous damage was demonstrated by the expression of 53BP1 and γ-H2AX foci （P<0.05） in all of the ovarian primary cells. After treatment with CPT and ionizing radiation, increased levels of DNA double-strand breaks were observed indicating a classic DNA damage response. Conclusion We have successfully established a protocol for the primary culture of epithelial ovarian cancer cells, which provides an important platform for characterizing DNA damage responses of the cells. With the progression of epithelial ovarian cancers, the ATM checkpoint pathway is activated by endogenous DNA lesions. This signaling pathway can be further activated by CPT or X-ray irradiation, hampering the growth of tumor cells and further progression of cancers.