The target cells for the transforming mutations caused by high-risk human papillomavirus (HPV) infection could be the stem cells of the uterine cervical epithelium, generating particular cancer stem cells (CSCs). The aim of this study was to identify and characterize the CSCs from cervical-cancer-derived cell lines. The ability of SiHa, CaLo, and C-33A cell lines to efflux Hoechst 33342 was evaluated by flow cytometry and cells from the corresponding side populations (SPs) and nonside populations (NSPs) were analyzed for their cell-cycle status (pyronin Y) and their mRNA levels of ABC transporter family members (with qPCR). Specific markers (α6-integrin(bri)/CD71(dim), CK17) of normal epithelial stem cells were evaluated by flow cytometry. The biological properties of these cells were analyzed, including their colony heterogeneity, repopulation, and anchorage-independent colony formation. We identified SPs (around 3 %) in the SiHa and CaLo cell lines, more than 70 % of which were in G0 phase and strongly expressed ABC transporters (predominantly ABCG2 and ABCB1). The SP from CaLo cells showed an α6-integrin(bri)/CD(dim) pattern, whereas the SP from the SiHa cells showed an α6-integrin(-)/CD(dim) pattern. Recultured cells from the SPs of both cell lines generated both SPs and NSPs, and had higher clonogenic potential to form mainly holoclones and greater colony-forming efficiency under anchorage-independent growth conditions than the cells from the NSPs or total cell populations. Interestingly, we identified no SP in the HPV-uninfected C-33A cell line, and it did not express ABCG2 or other members of the ABC transporters (ABCB1, ABCC1, or ABCA3).