The use of CD133 as a marker for identifying and isolating colon cancer stem cells (CSCs) is controversial. In particular, it is not clear whether the pattern of expression is influenced by the tumor microenvironment. We analysed the expression of CD133 in the SW620 colon cancer cell line, which produces subpopulations with different levels of CD133. CD133(neg) and CD133(hi) SW620 cells were isolated and compared in hanging drop spheroid cultures. The expression of CD133 was examined under different culture conditions and following nutrient withdrawal. The results showed that SW620 cells express the CD133 antigen at variable levels. CD133(neg) and CD133(hi) SW620 cells can be separated by fluorescence-activated cell sorting and, when grown as adherent cells in liquid culture, are serially passaged without changes to the CD133 levels. However, the CD133 antigen is re-expressed when the CD133(neg) cells are deprived of nutrients or grown as non-adherent cells in spheroid cultures. Our observations indicate that the presence of the CD133 antigen does not identify a stem cell population of SW620 colorectal cancer cells. The expression of CD133 is dynamic and is modulated during certain cell culture conditions, suggesting that changes in expression may be mediated by the energy supply available to the cells.