We have investigated the association of the glucocorticoid receptor in S49 mouse lymphoma cells with the 90-kDa heat shock protein, HSP 90. Proteins were synthetically labeled by culturing cells with [35S]methionine and immunoadsorbed to protein A-Sepharose using monoclonal antireceptor antibody (BuGR-2). HSP 90 coimmunoadsorbed with the receptor when cytosol was incubated with antireceptor antibody for 1 h in the absence of molybdate, indicating that molybdate is not required for either formation or recovery of HSP 90-receptor complexes. Molybdate stabilized HSP 90-receptor complexes during prolonged incubation in vitro and rendered the complexes resistant to dissociation by 500 mM NaCl. In pulse-chase experiments, cells were incubated with [35S]methionine for 30 min (pulse label) and then cultured with excess unlabeled methionine (chase). At different times in the chase, cytosol was prepared from cells, and labeled proteins were immunoadsorbed by antireceptor antibody. The amount of labeled receptor recovered from cytosol decreased during the chase with a half-life of about 4 h. Labeled HSP 90 coimmunoadsorbed with the receptor at 4 h into the chase but not at the beginning of the chase or at 2 h into the chase, indicating that newly synthesized HSP 90 associated with the receptor in a time-dependent manner during the chase. Labeled HSP 90 did not associate with the receptor when dexamethasone was added to cells at the beginning of the chase. These findings support the hypothesis that HSP 90 associates with the glucocorticoid receptor in intact cells rather than in cytosol and indicates that dexamethasone promotes dissociation of HSP 90-receptor complexes in vivo.