The molecular chaperone Hsp90 interacts with unliganded steroid hormone receptors and regulates their activity. We have analyzed the function of yeast and mammalian Hsp90 in regulating the ability of the human estrogen receptor (ER) to bind ligands in vivo and in vitro. Using the yeast system, we show that the ER expressed in several different hsp82 mutant strains binds reduced amounts of the synthetic estrogen diethylstilbestrol compared to the wild type. This defect in hormone binding occurs without any significant change in the steady state levels of ER protein. To analyze the role of mammalian Hsp90, we synthesized the human ER in rabbit reticulocyte lysates containing geldanamycin, an Hsp90 inhibitor. At low concentrations of geldanamycin we observed reduced levels of hormone binding by the ER. At higher concentrations, we found reduced synthesis of the receptor. These data indicate that Hsp90 functions to maintain the ER in a high affinity hormone-binding conformation.