Recently Gadd45, a p53-regulated stress protein, has been implicated in the activation of a G2/M checkpoint after damage by UV radiation and alkylating agents. While inhibitory phosphorylation of Cdc2 and suppression of cyclin B1 levels are known to be involved in G2 delays after genotoxic stress, Gadd45 has now been found to directly inhibit the activity of Cdc2/Cyclin B1 complex, while it had no appreciable effect on Cdk2/Cyclin E activity even at very high levels of Gadd45. In contrast, p21 Cip1/Waf1 is an universal cdk/cyclin inhibitor and inhibited both of the cyclin complexes tested here. Gadd45 was also able to physically interact with Cdc2, but not Cyclin B1. Addition of Gadd45 to immunoprecipitated Cdc2/Cyclin B1 in vitro led to a dissociation of this complex, and thus may represent a new checkpoint mechanism whereby Cdc2/Cyclin B1 can be inhibited. With the use of an antisense approach, reduced Gadd45 expression attenuated the suppression of Cdc2/Cyclin B1 activity in UV-irradiated human cells. Taken together, these results implicate Gadd45 in the control of G2/M cell cycle progression after certain stresses.