p21(Cip1/WAF1) is the principle mediator of cell cycle arrest in response to DNA damage. p21 primarily mediates G(1) cell cycle arrest by inactivating G(1)-associated cyclin A- and cyclin E-containing cyclin/cdk complexes. In the present study we investigate the role of p21 in DNA damage-induced G(2) cell cycle arrest, particularly with respect to the G(2)-associated cyclin, cyclin B1. We demonstrate that cells lacking p21 or deficient in their ability to upregulate p21 are unable to mediate the downregulation of cyclin B1 in response to DNA damage as compared to wild-type cells. Decreased levels of cyclin B1 in response to DNA damage seen in wild-type cells is due to p21-mediated degradation of cyclin B1 as this can be inhibited by a proteasomal inhibitor. Cell cycle analysis reveals that p21-null cells are unable sustain G(2) cell cycle arrest and accumulate at greater than 4N DNA content. These results indicate that p21-mediated degradation of cyclin B1 in response to DNA damage is necessary for the maintenance of G(2) cell cycle arrest.