Sex determining region Y-box 2 (SOX2) is well known as one of the "sternness" factors and is often expressed in cancers including breast cancer. In this study, we developed a reporter system using fluorescent protein driven by the promoter for SOX2 gene to detect and isolate living SOX2-positive cells. Using this system, we determined that SOX2 promoter activities were well correlated with SOX2 mRNA expression levels in 5 breast cancer cell lines, and that the cell population with positive SOX2 promoter activity (pSp-T+) isolated from one of the 5 cell lines, MCF-7 cells, showed a high SOX2 protein expression and high sphere-forming activity compared with very low promoter activity (pSp-Tlow/-). The pSp-T(+)pbpulation expressed higher mRNA levels of several stemness-related genes such as CD44, ABCB1, NANOG and TWIST] than the pSp-Tlow/- population whereas the two populations expressed CD24 at similar levels. These results suggest that the cell population with SOX2 promoter activity contains cancer stem cell (CSC)-like cells which show different expression profiles from those of CSC-marker genes previously recognized in human breast cancers. (C) 2013 Elsevier Inc. All rights reserved.