Abstract p53-p21-cyclin-dependent kinase and p16INK4a-cyclin-dependent kinase pathways have parallel functions in preventing tumorigenesis. In cancer patients, tumor suppressor p53 is frequently inactivated through mutations, whereas p16INK4a is silenced through promoter methylation. However, the interaction between these two pathways is less well understood. Here, we report that p53 controls p16INK4a expression in a unique way. p53 deficiency led to up-regulation of p16INK4a in primary mouse embryonic fibroblasts, osteoblasts, and various mouse organs, and an increase in the p16INK4a promoter activity, without affecting the half-life of p16INK4a. Reconstitution of p53, but not mutant p53, restored the proper expression of p16INK4a. These results indicate that p53 is necessary in repressing p16INK4a expression. However, up-regulation of p53 in response to genotoxic stress or nutlin-3 treatment did not down-regulate p16INK4a. p53 did not repress the p16INK4a promoter activity either. These findings suggest that p53 has a necessary but not sufficient role in repressing p16INK4a expression. p16INK4a elevation in p53−/− cells is, at least partially, mediated by Ets1, a known positive regulator of p16INK4a, as p53 deficiency up-regulated Ets1 through protein stabilization and knockdown of Ets1 down-regulated p16INK4a expression in p53−/− mouse embryonic fibroblasts. These studies uncover a compensatory mechanism for the loss of p53 and provide a basis for targeting both p53 and p16INK4a in cancer therapy. (Mol Cancer Res 2009;7(3):354–63)