We have shown previously that wild type p53 can rapidly induce replicative senescence in EJ human bladder carcinoma cells lacking functional p53. A major effector of p53 functions is p21 Waf1/Cip1/Sdi1, a potent cyclin-dependent kinase inhibitor. p21 Waf1/Cip1/Sdi1 has been shown to be involved in both p53 dependent and independent control of cell proliferation, differentiation and death. To directly investigate the effects of p21 Waf1/Cip1/Sid1 in the p53 response observed in EJ tumor cells, we established p21 Waf1/Cip1/Sdi1 inducible lines using the tetracycline-regulatable vector system. p21 Waf1/Cip1/Sdi1 induction caused irreversible cell cycle arrest in both G1 and G2/M, and diminished Cdk2 kinase activity. In addition, p21 Waf1/Cip1/Sdi1 induction led to morphological alterations characteristic of cells undergoing replicative senescence with morphological, biochemical and ultrastructural markers of the senescent phenotype. Furthermore, sustained p21 Waf1/Cip1/Sdi1 induction sensitized EJ cells to apoptotic cell death induced by mitomycin C, a cross-linking DNA damaging agent. These findings support the function of p21 Waf1/Cip1/Sdi1 as an inducer of replicative senescence and a major mediator of this phenomenon in response to p53. Moreover, our results imply that therapeutic intervention in human cancers might be aimed at sustained elevation of p21 Waf1/Cip1/Sdi1 expression.