Glucocorticoid receptors are basally phosphorylated in the absence of hormone and become hyperphosphorylated after hormone treatment of intact cells. To determine the sequence of changes which the receptor undergoes following hormone binding, we analyzed the kinetics of receptor phosphorylation in WEHI-7 mouse thymoma cells and in stably transfected Chinese hamster ovary cells that overexpress the mouse receptor. No major differences were found between these two cell types. Cells were preincubated with 32P(i) and [35S] methionine to label the receptors metabolically. The phosphate content of the receptor protein was determined from the ratio of 32P to 35S in radioactive gel slices after immunopurification and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Hormone-induced increases in phosphorylation were seen as early as 5 min after adding hormone and persisted for 20 h. Analysis of newly formed cytosolic and nuclear-bound activated (DNA-binding) receptors showed that activation precedes hyperphosphorylation. Nonactivated receptors, both unliganded and hormone-liganded, also became hyperphosphorylated but more slowly than activated receptors. The rate of receptor dephosphorylation, determined by chasing with unlabeled P(i), was much slower than the rate of phosphorylation or of hormone dissociation and appeared to be slightly increased by agonists and by the antagonist RU486 (which does not cause hyperphosphorylation). Mutant WEHI-7 cells lacking cAMP-dependent protein kinase activity gave basal and hormone-induced receptor phosphorylation indistinguishable from wild type cells. We conclude that (a) the substrate for hormone-dependent hyperphosphorylation is the activated hormone-receptor complex; (b) most hyperphosphorylated receptors are recycled and reutilized in hyperphosphorylated form; (c) control of receptor phosphorylation may not be cell-specific; (d) cAMP-dependent protein kinase is not involved directly or indirectly in phosphorylating major sites on the receptor in vivo.