The p19ARF gene product responds to oncogenic stresses by interfering with the inhibitory effects of Mdm2 on p53, thus enhancing p53 activity and its antiproliferative functions. The absence of p19ARF in the mouse leads to early tumor susceptibility, presumably in part due to decreased p53 activity. To examine the tumorigenic cooperativity of p19ARF, Mdm2, and p53 in vivo, p19ARF-deficient mice were crossed first to p53-deficient mice and then to Mdm2 transgenic mice. The progeny were monitored for tumors. Cooperativity between p19ARF and p53 deficiencies in accelerating tumor formation was observed for most genotypes except p53-/- p19ARF-/- mice. p53-/- p19ARF-/- mice had a tumor incidence similar to p53-/- mice. In this context, tumor suppression by ARF appears to be primarily p53 dependent. The majority of the p19ARF+/- tumors deleted the wildtype p19ARF allele, in agreement with the previous studies, suggesting that p19ARF is a classic 'two hit' tumor suppressor. In a p53+/- background, however, all p19ARF+/- tumors retained a wildtype ARF allele and most also retained wildtype p53. In the second cross between p19ARF-deficient and Mdm2 transgenic mice, cooperativity in tumor incidence between Mdm2 overexpression and ARF deficiency was observed, consistent with the role of p19ARF in negatively regulating Mdm2 activity. These experiments further demonstrate in vivo the inter-relationships of the p19ARF-Mdm2-p53 signaling axis in tumor suppression.