The response of glucocorticoid receptors (GR) to heat was measured by the change in ligand binding activity both in control cells and in cells made tolerant to heat by a prior mild heat exposure. The study was prompted by earlier data showing that one of the heat shock proteins (HSP90) is an essential component of the GR complex and that treatment of mammalian cells with hydrocortisone induces resistance to heat damage. The GR rapidly loses binding activity after commencement of heating. There is a 50% loss of activity after 4 min at 45 degrees C, 8 min at 44 degrees C, or 17 min at 43 degrees C. The reduction in binding is due mainly to a reduction in affinity of binding to the ligand. The ability to bind glucocorticoid recovers quickly after heat treatment. Activity returns to levels 60-80% of normal by 2 h after a heat treatment that initially reduces binding to less than 20% of normal. However, complete restoration of binding activity takes approximately 3 days. The recovery of binding activity does not require protein synthesis. Pretreatment of cells with hydrocortisone, using conditions that induce heat resistance, reduces the activity to 10-20% of control, but residual receptors display a heat sensitivity similar to that of control cells. There was evidence for a limited degree of protection of GR from heat damage in thermotolerant cells.