The cellular events mediating the rapid, thyroid hormone-dependent modulation of membrane-bound, type II iodothyronine 5'-deiodinase were studied in dibutyryl cyclic AMP(bt2cAMP)-treated brain astrocytes. Unstimulated cells had undetectable type II 5'-deiodinating activity. Treating the cells with bt2cAMP and hydrocortisone induced enzyme expression by increasing transcription of the enzyme or an essential enzyme related protein(s), with steady-state levels of type II 5'-deiodinase attained after 8 h of bt2cAMP treatment. Glial cells grown in the absence of thyroid hormone had 10-15-fold higher levels of 5'-deiodinating activity than cells grown in the presence of serum. The increased type II 5'-deiodinating activity observed in serum-free cultures was due to a prolonged enzyme half-life with no change in the rate of enzyme synthesis. Addition of thyroxine or 3,3',5'-triiodothyronine to the serum-free culture medium resulted in a concentration-dependent fall in steady-state enzyme levels, with EC50 values of approximately 0.4 nM. 3,5,3'-Triiodothyronine was at least 100-fold less effective. Chloroquine, NH4Cl, tunicamycin, colchicine, and monodansylcadavarine had no effect on the t1/2 of the enzyme, while both carbonyl cynanide m-chlorophenylhydrazone and cytochalasins completely blocked the inactivation of the type II 5'-deiodinase. These data indicate that in glial cells, an intact actin-cytoskeleton is required for thyroid hormone to modulate the energy-dependent regulation of the half-life of the short-lived, membrane-bound enzyme, type II 5'-deiodinase.