Abstract: 【Abstracts】 Objective To determine the effect of cellular density on the separation and identification of cancer stem cells from human ovarian clear cell carcinoma cell line ES-2 and adenocarcinoma cell line A2780. Methods ES-2 and A2780 cells were cultured with human recombinant epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and bovine serum albumin and insulin in serum free medium. The cancer stem cells were obtained through serial passages. Changes in cell morphology, expressions of surface marker CD133 and CD44, and soft AGAR clone forming in the stem cells were examined under different cell density, either in serum-supplemented medium (SSM group) or in serum free medium (SFM group). Results Under the density of 2×10⁴ mL^-1, ES-2 cells survived in SFM, but did not form stem cells. When the density increased to 5×10⁴ mL^-1 or 1×10⁵ mL^-1, ES-2 cells survived in SFM, proliferated and formed stem cells. Compared with adherent cells, the suspension globe of stem cells expressed high levels of CD133 and CD44 ( P<0.05), with proliferation and clone forming ability after serial passages. The stem cell balls under the density of 5×10⁴ mL^-1 had stronger ability of tumor formation. A2780 cells formed suspension globe under the density of 1×10⁴ mL^-1 and 3×10⁴ mL^-1, but larger and more transparent balls were observed under the density of 3×10⁴ mL^-1 density. No suspension globe was formed under the density of 5×10⁴ mL^-1. More CD133⁺/CD44⁺cells were detected by flow cytometry under the density of 3×10⁴ mL^-1, compared with that under the density of 1×10⁴ mL^-1 ( P<0.05). Tumor stem cells grew faster under the density of 3×10⁴ mL^-1. Conclusion The optimal density for identifying stem cells from human ovarian cancer is 5×10⁴ mL^-1 for ES-2 and 3×10⁴ mL^-1 for A2780, respectively.