The INK4a tumor suppressor locus encodes p16 ^INK4a, an inhibitor of cyclin D-dependent kinases, and p19 ^ARF, an alternative reading frame protein that also blocks cell proliferation. Surprisingly, mice lacking p19 ^ARF but expressing functional p16 ^INK4a develop tumors early in life. Their embryo fibroblasts (MEFs) do not senesce and are transformed by oncogenic Ha-ras alone. Conversion of ARF^+/+ or ARF^+/− MEF strains to continuously proliferating cell lines involves loss of either p19 ^ARF or p53. p53-mediated checkpoint control is unperturbed in ARF-null fibroblast strains, whereas p53-negative cell lines are resistant to p19 ^ARF-induced growth arrest. Therefore, INK4a encodes growth inhibitory proteins that act upstream of the retinoblastoma protein and p53. Mutations and deletions targeting this locus in cancer cells are unlikely to be functionally equivalent.