The p16^INK4 (MTS1) gene has many features of a tumor suppressor gene. It maps to 9p21, a region of frequent loss of heterozygosity in a variety of tumor types. It encodes an inhibitor of cyclin-dependent kinase 4, and its homozygous deletion is common in tumor-derived cell lines. To examine its tumor suppressive function and its potential in cancer gene replacement therapy, wild-type p16^INK4 was expressed in an adenovirus-derived gene delivery system and introduced into lung cancer cell lines that do not express p16^INK4. Expression of the introduced p16^INK4 blocked tumor cell entry into S phase of the cell cycle and inhibited tumor proliferation both in vitro and in vivo. These observations strongly support that p16^INK4 is a tumor suppressor gene and is a candidate for cancer gene replacement therapy.