The 90-kDa heat shock protein Hsp90 represents a highly conserved strongly expressed gene family; in Saccharomyces cerevisiae, Hsp90 proteins are essential for cell viability. Hsp90 interacts with certain cellular proteins, including steroid hormone receptors, tyrosine and serine/threonine kinases, and other heat shock proteins, but its biological functions are not understood. The unliganded glucocorticoid receptor must interact with Hsp90 to acquire competence for high-affinity hormone binding and subsequent transcriptional regulation. By screening in yeast for defects in glucocorticoid receptor function, Hsp90 mutants were isolated. Four such mutants are described, all of which interact with the glucocorticoid receptor but display distinct defects in ligand responsiveness and differences in growth and resistance to high temperature.