To better understand the mechanism(s) underlying lung cancer invasion and metastasis, a Transwell invasion chamber was used to select progressively more invasive cancer cell populations from a clonal cell line of human lung adenocarcinoma, CL1. Five sublines with progressive invasiveness, designated CL1-1, CL1-2, CL1-3, CL1-4, and CL1-5, were obtained through this in vitro selection process. Their invasive abilities through basement membrane matrix showed a 4- to 6-fold increase over that of the parental cells. Moreover, the sublines manifested an increase in their colony-forming ability on soft agar, tumorigenicity, and metastatic potency in severe combined immunodeficiency (SCID) mice. Examining the phenotypes of the cell lines revealed increased expression of 92 kD gelatinase and an increase in the cell population stained with anti-keratin-8 and -18 antibodies. Clonal isolation of anti-keratin-18-antibody-positive and -negative cell populations demonstrated a correlated enhancement of the invasiveness of these cells and their expression of keratin-18. These results support the notion that the metastatic behavior of lung cancer cells can be characterized with this in vitro system, and that the properties of these progressively invasive cancer cells can be clonally studied.