Background and purpose: CD133 is controversially discussed as putative (surrogate) marker for cancer stem/tumor-initiating cell populations (CSC/TIC) in epithelial tumors including colorectal carcinomas (CRCs). We studied CD133 expression in established CRC cell lines and examined in vitro behavior, radioresponse and in vivo tumor formation of CD133^+/- subpopulations of one cell line of interest. Materials and methods: Ten CRC cell lines were analyzed for CD133 expression using flow cytometry and Western blotting. CD133⁺ and CD133^- HCT-116 subpopulations were separated by FACS and studied in 2-D and 3-D culture and colony formation assays after irradiation. Subcutaneous xenograft formation was monitored in NMRI (nu/nu) mice. Results and conclusions: CRC cell lines could be classified into three groups: (i) CD133^-, (ii) CD133⁺ and (iii) those with two distinct CD133⁺ and CD133^- subpopulations. Isolated CD133^+/- HCT-116 subpopulations were studied relative to the original fraction. No difference was found in 2-D growth, spheroid formation or radioresponse in vitro. Also, tumor formation and growth rate did not differ for the sorted subpopulations. However, a subset of xenografts originated from CD133^- HCT-116 showed a striking enrichment in the CD133⁺ fraction. Our data show that CD133 expression is not selective for sphere forming, tumor-initiating or radioresistant subpopulations in the HCT-116 CRC cell line. This implies that CD133 cannot be regarded as a CSC/TIC marker in all CRC cell lines and that functional measurements of tumor formation have to generally accompany CSC/TIC-directed mechanistic or therapeutic studies.