Cancer stem cells (CSCs) have been identified in solid tumors and cancer cell lines. In this study, we isolated a series of cancer cell clones, which were heterogeneous in growth rate, cell cycle distribution and expression profile of genes and proteins, from ovarian tumor specimens of a patient and identified a sub-population enriched for ovarian CSCs defined by CD24 phenotype. Experiments in vitro demonstrated CD24(+) sub-population possessed stem cell-like characteristics of remaining quiescence and more chemoresistant compared with CD24(-) fraction, as well as a specific capacity for self-renewal and differentiation. In addition, injection of 5 x 10(3) CD24(+) cells was able to form tumor xenografts in nude mice, whereas equal number of CD24(-) cells remained nontumorigenic. We also found that CD24(+) cells expressed higher mRNA levels of some 'stemness' genes, including Nestin, beta-catenin, Bmi-1, Oct4, Oct3/4, Notch1 and Notch4 which were involved in modulating many functions of stem cells, and lower E-cadherin mRNA level than CD24(-) cells. Altogether, these observations suggest human ovarian tumor cells are organized as a hierarchy and CD24 demarcates an ovarian cancer-initiating cell population. These findings will have important clinical applications for developing effective therapeutic strategies to treat ovarian cancer.