The activity and structural integrity of the tumor suppressor protein p53 is of crucial importance for the prevention of cancer. p53 is a conformational flexible and labile protein, in which structured and unstructured regions function in a synergistic manner. The molecular chaperone Hsp90 is known to bind to mutant and wild type p53 in vivo. Using highly purified proteins we analyzed the interaction and the binding sites between both proteins in detail. Our results demonstrate that Hsp90 binds to a folded, native-like conformation of p53 in vitro with micromolar affinity. Specifically, the DNA-binding domain of p53 and the middle and carboxy-terminal domains of Hsp90 are responsible for this interaction, which is essential to stabilize p53 at physiological temperatures and to prevent it from irreversible thermal inactivation. Our results are in agreement with a model in which Hsp90 is required to maintain the folded, active state of p53 by a reversible interaction, thus introducing an additional level of regulation.