Mice heterozygous for the retinoblastoma (Rb) tumor suppressor gene develop pituitary and thyroid tumors with high penetrance. We demonstrate here that loss of the ARF tumor suppressor strongly accelerates intermediate lobe pituitary tumorigenesis in Rb heterozygous mice. These effects in the pituitary are greater than those conferred by p53 loss in that Rb+-;ARF-- mice display significantly more early atypical lesions than Rb+-; p53-- mice. Also, Rb+-;ARF-- compound mutants do not develop many of the novel tumors or precancerous lesions seen in Rb+-;p53-- compound mutants. Although complete loss of ARF expression is not obligatory for pituitary tumorigenesis in Rb+- mice, alterations of the ARF locus are observed in tumors from Rb+-;ARF+- mice, consistent with a selective advantage of ARF inactivation in this context. We conclude that inactivation of ARF acts more broadly than that of p53 in connecting abrogation of the Rb pathway to tumorigenesis.