Cell cycle arrest represents an important response to genotoxic stress and the tumor suppressor p53 has been described to act as a critical effector in this biological event. Upon stress, p53 becomes transcriptionally active and up-regulates the transcription of downstream effector genes, which contain p53 recognition sites in their regulatory regions. Among the genes activated are p21 and GADD45, each of which independently exhibits growth-suppressive activity. The Gadd45 protein has been described to form a complex with p21, and thus, work was undertaken to map the regions of Gadd45 involved in this interaction and to examine the roles of those two proteins in growth suppression. In this report, a Gadd45 overlapping peptide library and a series of Gadd45 deletion mutants were used to define the domains of Gadd45 involved in the association with p21. Results using both in vitro and in vivo methods have shown that the interaction of Gadd45 with p21 involves a central region of Gadd45. Interestingly, the p21-binding domain of Gadd45 also encodes the Cdc2-binding activity, indicating that the central region of Gadd45 may serve as an important "core," through which Gadd45 protein is able to present cross-talk with other cell cycle regulators. In addition, GADD45 inhibition of Cdc2 kinase activity was compared with Myd118 and CR6, two other members of the GADD45 family. GADD45 was shown to generate the strongest inhibitory effect on Cdc2 activity. Finally, results from short-term survival assays further demonstrated that p21 and GADD45 act upon different cellular pathways to exert their growth-suppressive function.