In the brain, thyroid hormone dynamically regulates levels of the short-lived plasma membrane protein, type II iodothyronine 5'-deiodinase. In cultured astrocytes, thyroxine modulates deiodinase levels by activating cytoskeletal-plasma membrane interactions that increase the rate of inactivation of the enzyme. Here we characterized the effects of these thyroxine-dependent cytoskeletal interactions upon the route of internalization of the deiodinase by following the intracellular transit of the affinity-labeled substrate-binding subunit of the deiodinase (p29). Thyroxine rapidly induced the inactivation of the deiodinase and initiated the binding of p29 to F-actin. By 40 min, > 75% of the p29 had been transported to an endosomal pool, which was followed by dissociation of the F-actin-p29 complex. There was no significant accumulation of p29 in the dense lysosomes seen in the presence of thyroxine. In the absence of thyroxine, p29 was internalized and transported to the dense lysosomes at a rate parallel to the inactivation rate of the deiodinase (t1/2 0.75 and 0.64 h, respectively) without involvement with the microfilaments. These data demonstrate that thyroxine targets type II iodothyronine 5'-deiodinase to an endosomal pool by activating specific protein-F-actin interactions involved in microfilament-mediated intracellular protein trafficking.