To investigate whether aldehyde dehydrogenase-1 (ALDH-1) in human lung cancer can be used as a sorting marker for stem cells in targeted therapies against human lung cancer. Spheres were induced by incubating cancer cells in a serum-free medium and formed with epidermal growth factor and fibroblast growth factor-10 (FGF10). Spheroid cells were combined with flow cytometry using the Aldefluor reagent to separate the SSCloALDEbr (ALDH-1-positive) cells. Cancer stem cells (CSCs) were characterized by their proliferation, colony formation, and tumorigenesis in nude mice and using phenotypic analysis. Float-growing spheres (“pulmospheres”) were developed after SPC-A1 cells were cultured in a serum-free medium. The resultant sphere-forming cells included ALDH-1-positive cells as high as 15.13%. ALDH-1-positive CSCs have high proliferative ability, high cloning efficiency, and strong tumorigenicity. The rate of SSCloALDEbr cell colony formation was 1.3–5.6%, whereas that of ALDE− cell colony formation was only 0–1.2% (P < 0.05). A cell count of only 1 × 103 SSCloALDEbr cells was necessary to form tumors, whereas at least l × 105 ALDE− cells formed tumors. The same number of SSCloALDEbr cells also formed larger tumors in a short latency period of tumor formation. The expression rates of CD133 in the SSCloALDEbr and ALDE− cells were 16.31% (16.31 × 104/106) and 2.56% (2.56 × 104/106), respectively (P < 0.01). Moreover, the expression rates of ABCG2 in SSCloALDEbr and ALDE− cells were 17.62% (17.62 × 104/106) and 3.45% (3.45 × 104/106), respectively (P < 0.01). Human lung adenocarcinoma bears CSCs, and ALDH-1 can act as a specific marker for human lung CSCs.