The cancer stem cell (CSC) model, in which a small population of cells within a tumor possesses the ability to self-renew and reconstitute the phenotype of primary tumor, has gained wide acceptance based on evidence over the past decade. It has also been reported that cancer cell lines contain a CSC subpopulation. However, phenotypic differences between CSCs and non-CSCs in cancer cell lines are not better defined than in primary tumors. Furthermore, some cell lines do not have a CSC population, revealed as a side population and expression of CD133. Thus, the identification of CSCs in cancer cell lines remains elusive. Here, we investigated the CSC hierarchy within HCT116 colon cancer cells, which do not have a CD133-positive subpopulation. We examined the expression of alternative CSC markers epithelial specific antigen (ESA) and CD44 in floating-sphere-derived cells, which are known to be the cells of enriching CSCs. Sphere-derived HCT116 cells exhibited heterogeneous expression of ESA and CD44. The two major subpopulations of HCT116 sphere cells (ESAlowCD44−/low and ESAhighCD44high) exhibited a biological/proliferative hierarchy of sphere-forming and soft agar colony-forming activity. However, there was no difference between the two subpopulations in the incidence of xenograft tumors. When ESAlowCD44−/low cells were allowed to aggregate and re-form floating-spheres, the biological/proliferative hierarchy of parental HCT116 spheres was reconstituted, in terms of ESA and CD44 expression. Thus, HCT116 cells have plasticity when they are set in floating-spheres, suggesting that maintenance of the HCT116 cell line conforms to a stochastic model, not a CSC model. (Cancer Sci 2009; 100: 2275–2282)