The commitment of cells to replicate and divide correlates with the activation of cyclin-dependent kinases and the inactivation of Rb, the product of the retinoblastoma tumor suppressor gene. Rb is a target of the cyclin-dependent kinases and, when phosphorylated, is inactivated. Biochemical studies exploring the nature of the relationship between cyclin-dependent kinase inhibitors and Rb have supported the hypothesis that these proteins are on a linear pathway regulating commitment. We have been able to study this relationship by genetic means by examining the phenotype of Rb+/-p27-/- mice. Tumors arise from the intermediate lobe cells of the pituitary gland in p27-/- mice, as well as in Rb+/- mice after loss of the remaining wild-type allele of Rb. Using these mouse models, we examined the genetic interaction between Rb and p27. We found that the development of pituitary tumors in Rb+/- mice correlated with a reduction in p27 mRNA and protein expression. To determine whether the loss of p27 was an indirect consequence of tumor formation or a contributing factor to the development of this tumor, we analyzed the phenotype of Rb+/-p27-/- mice. We found that these mice developed pituitary adenocarcinoma with loss of the remaining wild-type allele of Rb and a high-grade thyroid C cell carcinoma that was more aggressive than the disease in either Rb+/- or p27-/- mice. Importantly, we detected both pituitary and thyroid tumors earlier in the Rb+/-p27-/- mice. We therefore propose that Rb and p27 cooperate to suppress tumor development by integrating different regulatory signals.