One emerging model for the development of drug-resistant tumors utilizes a pool of self-renewing malignant progenitors known as cancer stem cells (CSCs) or cancer-initiating cells (CICs). The purpose of this study was to propagate such CICs from the ovarian cancer cell line SKOV3. The SKOV3 sphere cells were selected using 40.0 micromol/l cisplatin and 10.0 micromol/l paclitaxel in serum-free culture system supplemented with epidermal growth factor, basic fibroblast growth factor, leukemia inhibitory factor, and insulin or standard serum-containing system. These cells formed non-adherent spheres under drug selection (cisplatin and paclitaxel) and serum-free culture system. The selected sphere cells are more resistant to cisplatin, paclitaxel, adriamycin, and methotrexate. Importantly, the sphere cells have the properties of self-renewal, with high expression of the stem cell genes Nanog, Oct4, sox2, nestin, ABCG2, CD133, and the stem cell factor receptor CD117 (c-kit). Consistently, flow cytometric analysis revealed that the sphere cells have a much higher percentage of CD133(+)/CD117(+)-positive cells (71%) than differentiated cells (33%). Moreover, the SKOV3 sphere cells are more tumorigenic. Furthermore, cDNA microarray and subsequent ontological analyses revealed that a large proportion of the classified genes were related to angiogenesis, extracellular matrix, integrin-mediated signaling pathway, cell adhesion, and cell proliferation. The subpopulation isolation from the SKOV3 cell line under this culture system offers a suitable in vitro model for studying ovarian CSCs in terms of their survival, self-renewal, and chemoresistance, and for developing therapeutic drugs that specifically interfere with ovarian CSCs.