The cell cycle inhibitor p16^INK4a is inactivated in many human tumors and in families with hereditary melanoma and pancreatic cancer. Tumor-associated alterations in the INK4a locus may also affect the overlapping gene encoding p19^ARF and the adjacent gene encoding p15^INK4b, both negative regulators of cell proliferation. We report the phenotype of mice carrying a targeted deletion of the INK4a locus that eliminates both p16^INK4a and p19^ARF. The mice are viable but develop spontaneous tumors at an early age and are highly sensitive to carcinogenic treatments. INK4a-deficient primary fibroblasts proliferate rapidly and have a high colony-formation efficiency. In contrast with normal cells, the introduction of activated Ha-ras into INK4a-deficient fibroblasts can result in neoplastic transformation. These findings directly demonstrate that the INK4a locus functions to suppress neoplastic growth.