Mounting genetic evidence suggests that each product of the Ink4a/Arf locus, p16 INK4a and p19 ARF, possesses tumor-suppressor activity (Kamijo et al., 1997; Krimpenfort et al., 2001; Sharpless et al., 2001a). We report the generation and characterization of a p19 ARF-specific knockout allele (p19 ARF−/−) and direct comparison with mice and derivative cells deficient for p16 INK4a, both p16 INK4a and p19 ARF, and p53. Like Ink4a/Arf−/− murine embryo fibroblasts (MEFs), p19 ARF−/− MEFs were highly susceptible to oncogenic transformation, exhibited enhanced subcloning efficiency at low density, and resisted both RAS-and culture-induced growth arrest. In contrast, the biological profile of p16 INK4a−/− MEFs in these assays more closely resembled that of wild-type cells. In vivo, however, both p19 ARF−/− and p16 INK4a−/− animals were significantly more tumor prone than wild-type animals, but each less so than p53−/− or Ink4a/Arf−/− animals, and with differing tumor spectra. These data confirm the predominant role of p19 ARF over p16 INK4a in cell culture-based assays of MEFs, yet also underscore the importance of the analysis of tumor suppressors across many cell types within the organism. The cancer-prone conditions of mice singly deficient for either p16 INK4a or p19 ARF agree with data derived from human cancer genetics, and reinforce the view that both gene products play significant and nonredundant roles in suppressing malignant transformation in vivo.