In human gliomas, self‐renewing and tumor‐initiating cells are characterized by the expression marker CD133. Although, widely used, the validity of CD133 is debated as recent data show that CD133⁺ and CD133⁻ cells share similar stemness and tumorigenic properties. To clarify this “CD133 controversy”, we reexamined the methods of purification and the stem behavior of both CD133 compartments in fresh gliomas and gliomasphere cultures. Using human anti‐CD133‐coupled microbeads and magnetic activated cell sorting, we observed a nonspecific sorting of glioma cells irrespective of their CD133 expression. In contrast, when purified by fluorescence activating cell sorting, a specific expression and enrichment of CD133 was successfully observed in fresh human gliomas and gliomasphere cultures. However, neither the expression of stemness genes nor the long‐term self‐renewal capacities of CD133⁺ and CD133⁻ cells were significantly different, even after fresh isolation. Altogether, our data show that purification of CD133⁺ glioma cells using hCD133‐microbeads presents a lack of specificity and demonstrate that the use of CD133 as a unique glioma stem cell marker is likely not sufficient to tag the whole self‐renewing tumor cell reservoir. © 2009 UICC