Irreversible G 1 arrest in senescent human fibroblasts is mediated by two inhibitors of cyclin-dependent kinases (Cdks), p21Cip1/SDI1/WAF1 and p16 Ink4A . To determine the physiological and molecular events that specifically require p21, we studied senescence in human diploid fibroblasts expressing the human papillomavirus type 16 E6 oncogene, which confers low p21 levels via enhanced p53 degradation. We show that in late-passage E6 cells, high Cdk activity drives the cell cycle, but population expansion is slowed down by crisis-like events, probably owing to defective cell cycle checkpoints. At the end of lifespan, terminal-passage E6 cells exhibited several aspects of the senescent phenotype and accumulated unphosphorylated pRb and p16. However, both replication and cyclin-Cdk2 kinase activity were still not blocked, demonstrating that phenotypic and replicative senescence are uncoupled in the absence of normal p21 levels. At this stage, E6 cells also failed to upregulate p27 and inactivate cyclin-Cdk complexes in response to serum deprivation. Eventually, irreversible G 1 arrest occurred coincident with inactivation of cyclin E-Cdk2 owing to association with p21. Similarly, when p21 ؊/؊ mouse embryo fibroblasts reached the end of their lifespan, they had the appearance of senescent cells yet, in contrast to their wild-type counterparts, they were deficient in downregulating bromodeoxyuridine incorporation, cyclin E-and cyclin A-Cdk2 activity, and inhibiting pRb hyperphosphorylation. These data support the model that the critical event ensuring G 1 arrest in senescence is p21-dependent Cdk inactivation, while other aspects of senescent phenotype appear to occur independently of p21.Human diploid fibroblasts (HDFs) have a finite proliferative lifespan, at the end of which they cease irreversibly to divide and they undergo a series of phenotypic changes that distinguish senescence from quiescence (26). These phenotypic changes include altered morphology, increased cell volume, expression of a neutral senescence-associated ␤-galactosidase activity (SA-␤-Gal), and increased production of extracellular matrix degradative enzymes such as collagenase and stromelysin (26,40,61). It is now generally accepted that two inhibitors of cyclin-dependent kinases (Cdks), p16Ink4a (p16) and p21Cip1/ Waf1/Sdi1 (p21), whose amounts increase with age, have an essential role in inactivating Cdks in senescent fibroblasts (1,24,42,44,60). Cdk inactivation, in turn, allows the accumulation of unphosphorylated retinoblastoma protein (pRb) (59), a growth suppressor whose function is modulated by Cdks. Unphosphorylated pRb exerts negative regulation of cell cycle progression by forming complexes with members of the E2F transcription factor family (23, 28). In spite of their undisputed role in mediating senescence, the precise contribution of each Cdk inhibitor (CKI) is not fully established. The CKI p21 binds to and inactivates most cyclin-Cdk complexes, whereas p16 blocks cyclin D-Cdk activation by binding specifically to Cdk4 and Cdk6,...