Thyroid hormone, specifically thyroxine, alters cytoskeletal organization in astrocytes by modulating actin polymerization and, in turn, regulates the turnover of the short-lived membrane protein, type II iodothyronine 5'-deiodinase. In the absence of thyroxine, approximately 35% of the total cellular actin is depolymerized, and greater than 90% of the deiodinase is found in the plasma membrane and not associated with the cytoskeleton. Addition of thyroxine promotes actin polymerization and decreases the depolymerized actin to approximately 10% of the total actin pool, induces binding of the deiodinase to F-actin, and promotes rapid internalization of the enzyme. These data provide direct evidence that the actin cytoskeleton participates in the inactivation pathway of the deiodinase by translocating this short-lived plasma membrane protein to an internal membrane pool.