Objective To study whether the primary ovarian cancer cells containing cancer stem cells and its characterization in serum-free culture condition.Methods The primary cancer cells were isolated from one stage Ⅲ, grade 2 serous adenocareinoma tissue.Cells were cultured in serum-free culture system supplemented with epidermal growth factor,basic fibroblast growth factor,leukemia inhibitory factor and insulin.or standard serum-containing system.Methyl thiazolyl tetrazolium assay,quantitative PCR analysis,flow-cytometric analysis and xenograft experiments in vivo were performed.Results The primary cancer cells could maintain and forill cell sphere in serum-free culture system.These cells had the properties of self-renewal,overexpression of stem cell marker genes Nanog,Oct-4,Sox-2,nestin,ABCG2,CD_(133) and CD_(117) By contrast with the difierentiated cells under standard serum-containing culture conditions.these sphere-forming cells were more resistant to cisplatin and paclitaxel after treated 48 and 72 hours(61% vs.31%,73% vs.29%,P<0.05).With Hoechst 33342 exclusion assay,only 21.83%of sphere-forming cells were positive with the dye,compared with 83.04% positive cells in differentiated cells (P<0.01).Only 500 sphere-forming cells resulted subcutaneous xenograft tumors.All of these xenografts were categorized as serous adenocareinomas, overexpression of CA_(125) and cytokeratin-7 which were original tumor phenotype of ovarian cancer. Conclusion The sphere-forming cells isolated from primary ovarian cancer tissues have the characterization of cancer stem cell and may be a more reliable model system for understanding the biology of primary human tumors.