AIM: To identify cancer stem cells (CSCs) in human gallbladder carcinomas (GBCs). METHODS: Primary GBC cells were cultured under serum-free conditions to produce floating spheres. The stem-cell properties of the sphere-forming cells, including self-renewal, differentiation potential, chemoresistance and tumorigenicity, were determined in vitro or in vivo. Cell surface expression of CD133 was investigated in primary tumors and in spheroid cells using flow cytometry. The sphere-colony-formation ability and tumorigenicity of CD133(+) cells were assayed. RESULTS: In vitro culture experiments revealed that floating spheroids were generated from primary GBC cells, and these sphere-forming cells could generate new progeny spheroids in serum-free media. Spheroid cells were differentiated under serum-containing conditions with downregulation of the stem cell markers Oct-4, Nanog, and nestin (P < 0.05). The differentiated cells showed lower spheroid-colony-formation ability than the original spheroid cells (P < 0.05). Spheroid cells were more resistant to chemotherapeutic reagents than the congenetic adherent cells (P < 0.05). Flow cytometry showed enriched CD133(+) population in sphere-forming cells (P < 0.05). CD133(+) cells possessed high colony-formation ability than the CD133(-) population (P < 0.01). CD133(+) cells injected into nude mice revealed higher tumorigenicity than their antigen-negative counterparts (P < 0.05). CONCLUSION: CD133 may be a cell surface marker for CSCs in GBC. (C) 2011 Baishideng. All rights reserved.