Osteosarcoma is an aggressive primary bone cancer affecting primarily children and young adolescents. Development of valuable diagnostic indicators and therapeutic agents will be enhanced by the identification and characterization of genes that contribute to its aggressive behavior. Emerging evidence suggests a subpopulation of cancer stem cells within tumors that have been implicated in the pathogenesis of heterogeneous malignant tumors. The purpose of our study was to characterize the stem-like cell population in human osteosarcoma. Four human osteosacoma cell lines were cultured using a previously developed sarcosphere formation assay. The results showed that all human osteosarcoma cell lines possessed an ability to form sarcospheres. More spherical and frequent sarcospheres were observed in OS99-1 and MG63 cells than in Hu09 and Saos-2 cells. Moreover, stem cell markers Oct3/4 and Nanog were examined in osteosarcoma cell lines using RT-PCR and immunological techniques. For the first time, we showed that 2 Oct3/4 isoforms, Oct3/4 A and Oct3/4 B, were expressed in all 4 cell lines although they illustrated different expression patterns. Oct3/4 A was expressed in the nuclei while Oct3/4 B was located in the cytoplasm of a subpopulation of cells found within all four cell lines. Furthermore, elevated expression of Oct3/4 A was seen in the OS99-1, Hu09, and MG63 cells compared to Saos-2 cells while significantly higher expression of Oct3/4 B was detected in Hu09 compared with the other cell lines. In addition, Nanog was detected in a subpopulation of all cell lines, where OS99-1 cells expressed significantly higher levels than Hu09, Saos-2 and MG63 cells. Thus, our data support the cancer stem cell hypothesis, which may have important implications for clinic diagnosis and treatment of osteosarcoma.