The recent discovery of direct interactions between two important regulators of cell fate, the tumor suppressor p53 and glycogen synthase kinase-3b (GSK3b), led us to examine the mechanism and outcomes of this interaction. Two regions of p53 were identified that regulate its binding to GSK3b. Deletion of the p53 activation domain-1 (AD1), but not mutations that prevent MDM2 binding through the AD1 domain, enhanced GSK3b binding to p53, indicating that the AD1 domain interferes with p53 binding to GSK3b. Deletion of the p53 basic domain (BD) abrogated GSK3b binding, and a ten amino acid region within the C-terminal BD domain was identified as necessary for binding to GSK3b. GSK3b activity was not required for p53 binding, but inhibition of GSK3b stabilized the association, suggesting a transient interaction during which active GSK3b promotes actions of p53. This regulatory role of GSK3b was demonstrated by large reductions of p53-induced increases in the levels of MDM2, p21, and Bax when GSK3b was inhibited. Besides promoting p53-mediated transcription, GSK3b also contributed to mitochondrial p53 apoptotic signaling. After DNA damage, mitochondrial GSK3b co-immunoprecipitated with p53 and was activated, and inhibition of GSK3b blocked cytochrome c release and caspase-3 activation. Thus, GSK3b interacts with p53 in both the nucleus and mitochondria and promotes its actions at both sites.