Objective: To separate/enrich tumor stem-like cells from the human esophageal carcinoma cell line OE-19 by using serum-free suspension culture and to identify their biological characteristics and radiation resistance. Methods: OE-19 cells were cultivated using adherent and suspension culture methods. The tumor stem-like phenotype of CD44 expression was detected using flow cytometry. We examined growth characteristics, cloning capacity in soft agar, and radiation resistance of 2 groups of cells. Results: Suspended cells in serum-free medium formed spheres that were enriched for CD44 expression. CD44 was expressed in 62.5% of suspended cells, but only in 11.7% of adherent cells. The suspended cells had greater capacity for proliferation and colony formation in soft agar than the adherent cells. When the suspended and adherent cells were irradiated at 5 Gy, 10 Gy, or 15 Gy, the proportion of CD44+ suspended cells strongly and weakly positive for CD44 was 77.8%, 66.5%, 57.5%; and 21.7%, 31.6%, 41.4%, respectively. In contrast, the proportion of CD44+ adherent cells strongly positive for CD44 was 18.9%, 14.%, and 9.95%, respectively. When the irradiation dose was increased to 30 Gy, the survival of the suspended and adherent cells was significantly reduced, and viable CD44+ cells were not detected. Conclusion: Suspended cell spheres generated from OE-19 esophageal carcinoma cells in serum-free stem medium are enriched in tumor stem-like cells. CD44 may be a marker for these cells.