Abstract: Objective : To isolate cell spheres containing cancer stem cells (CSCs) from lung cancer cell lines (A 549 and H1299) and iden-tify their biological characteristics. Methods:By adopting the method of serum- free suspension culture, A549 cells and H 1299cells were cultured on no-adhesion plate to form tumor spheres. Clone assay, CCK-8 assay, and Transwell assay were employed to observe proliferation, self- renewal and invasion of tumor spheres. Besides, RT- PCR was performed to compare the expression levels of stem cell markers between sphere cells and adherent cells. Adherent A549 cells and A 549 cell spheres were inoculated subcutaneously in nude mice and the tumor growth was assessed. Results:Isolated CSCs from A 549 cells and H 1299cells in serum-free medium (SFM) without adhesion could grow as floating cell spheres. The results demonstrated that the self- renewal, proliferation and invasion of A549 and H1299sphere cells were stronger than parent cells (P<0. 05). When compared with adherent cells, the mRNA level of expres -sion of cell spheres' stem cell markers (Oct4 and Nanog) were significantly high ( P<0. 05) and A 549 spheres had a stronger tumorigenici -ty in nude mice. Conclusion: SFM without adhesion can be a quick and easy method to construct stem cells model from human lung adenocarcinoma cell lines A549 and H1299.