Abnormalities in the p16INK4a tumor suppressor gene are found in many lung cancer cell lines and primary lung cancer tissue. To examine its tumor suppressor function and potential adequacy in cancer gene replacement therapy, wild-type p16INK4a gene was inserted in an adenovirus derived gene delivery system and introduced into lung cancer cell lines (NCI-H441 and NCI-H157) that did not express p16INK4a. Western blot assay and immunocytochemistry demonstrated production of wild-type p16 protein in these cell lines. The biological function of exogenous p16 protein was confirmed by the inhibition of pRB phosphorylation. The expression of exogenous p16 protein via recombinant adenovirus significantly inhibited cancer cell growth and colony formation in vitro of NSCLC that can not express endogenous p16. The flow cytometric analysis showed these results correlated with G1 cell cycle arrest. These observations suggest the value of adenovirally-mediated p16INK4a gene replacement therapy for lung cancer.