The activity of the tumor suppressor p53 has to be timed and balanced closely to prevent untimely induction of cell death. The stability of p53 depends on the ubiquitin ligase Mdm2 but also on Hsp70 and Hsp90 chaperones that interact with its DNA binding domain (DBD). Using hydrogen exchange mass spectrometry and biochemical methods, we analyzed conformational states of wild-type p53-DBD at physiological temperatures and conformational perturbations in three frequent p53 cancer mutants. We demonstrate that the Hsp70/Hdj1 system shifts the conformational equilibrium of p53 toward a flexible, more mutant-like, DNA binding inactive state by binding to the DNA binding loop. The analyzed cancer mutants are likewise destabilized by interaction with the Hsp70/Hdj1 system. In contrast, Hsp90 protects the DBD of p53 wild-type and mutant proteins from unfolding. We propose that the Hsp70 and Hsp90 chaperone systems assume complementary functions to optimally balance conformational plasticity with conformational stability. Display omitted •Hsp70/Hdj1 increase local unfolding in wild-type and mutant p53-DNA binding domain•Hsp70/Hdj1 dissociates p53 from DNA by binding to its DNA binding loop•Hsp90 counteracts unfolding by Hsp70/Hdj1 and globally stabilizes p53•Hsp70 and Hsp90 balance p53 transcriptional activity by regulating its conformation The transcriptional activity of the tumor suppressor p53, which is essential to eliminate lethally damaged and oncogenic transformed cells, must be timed and balanced closely to prevent untimely induction of cell death. Boysen et al. show that Hsp70 and Hsp90 regulate p53’s conformation and DNA binding activity in opposing directions.