Abstract: Background and objective. Tumor heterogeneity has been shown to be related to clinical outcomes in cancer patients. The concept of a small subset of cancer stem cells responsible for tumor relapse and metastasis appears to be a promising strategy for targeted cancer treatment. However, cancer stem cells are not easy to identify and isolate. The aim of this study was to determine the putative colon cancer stem cell subsets in the human colon cancer cell lines HCT116 and HT29, which differ in their aggressiveness and differentiation capacity. Material and methods. Flow cytometry was used to evaluate HCT116 and HT29 cell lines for expression of stem-associated surface markers CD24, CD44, CD117, CD133, ESA, ABCB1. Both cell lines were treated with 5-fluoruracil and the phenotype of chemoresistant cells was studied. The secondary population was visualized via Rhodamine 123 staining. The relative expression of ABCG2, c-Myc and Oct4 genes was quantified by qPCR analysis. Results and conclusions. The HCT116 and HT29 cell lines have been shown to differ in their stemness-related properties. We imply that the putative CSC subset for the HCT116 cell line is CD44+/CD24-/CD133- (4.1% of all cells) and for HT29 cells – CD24+/CD44-/CD133- (4.9 % of all cells). : tumor heterogeneity, colon cancer, cancer stem cells, HCT116, HT29IntroductionThe concept of tumor heterogeneity linked to disease progression and clinical outcomes in cancer patients attracts significant attention in the era of personalized medicine (1). Current cancer treatment strategies rely on the site of origin of the primary tumor. However, tumors developed from distinct cell types have been shown to differ in their prognosis and response to cytotoxic therapies (2...... middle of article......ozzi E, Biffoni M , Todaro M, Peschle C, et al. Identification and expansion of human colon cancer-initiating cells Nature 2007;445(7123):111-5.25, Butler JM, Hooper AT, Hormigo A, Kushner J, Milde T et al. expression is not restricted to stem cells, and metastatic CD133+ and CD133- colon cancer cells trigger tumors Lian L, Zou Y et al. A subpopulation of CD24(+) cells in cell lines. of colon cancer has stem cell characteristics 2012; 59 (3): 282-8.27. CD44 is of functional importance for colorectal cancer stem cells Clin Cancer Res 2008;14. (21):6751-60.28. Stem cell markers: a persistent ambiguity.. 2012.