3,3'5,5'-Tetraiodothyronine (T4), but not 3,3'5-triiodothyronine (T3), acutely regulates the activity of the plasma membrane-bound enzyme, type II iodothyronine 5'-deiodinase (5'D-II), by inducing internalization of the enzyme through an extranuclear, energy-dependent mechanism that requires an intact actin cytoskeleton. The affinity label, N-bromoacetyl-L-T4, binds to 5'D-II and irreversibly inhibits the enzyme but does not initiate internalization. To determine the structural elements of T4 which are required for enzyme internalization, T4 analogs were modified in the alanine side chain and were then evaluated for their ability to induce enzyme internalization, to inhibit enzyme activity, and to promote actin polymerization in hypothyroid cells. The analogs studied showed marked variability in their ability to inactivate 5'D-II. The rank order of potency for enzyme inactivation was T4 > COOH-blocked analogs > NH3 and COOH blocked analogs >> NH3 blocked analogs (EC50 values range from 1 to > 1000 nM). In contrast, all T4 analogs tested and T4 were excellent competitive inhibitors of 5'D-II with respect to substrate (Ki values ranged from 4 to 27 nM). The differential capability of iodothyronines to inactivate the enzyme was not related to their ability to enter the cell, since Ki values measured in intact glial cells were equivalent to those measured in cell sonicates. The power of the T4 analogs to inactivate 5'D-II was paralleled by their ability to polymerize actin in hypothyroid cells and to induce 5'D-II binding to F-actin. The data show that modification of the alanine side chain markedly alters the ability of T4 analogs to induce 5'D-II inactivation and actin polymerization. A net negative charge on the alanine side chain of T4 is detrimental for the hormone-dependent inactivation of 5'D-II and polymerization of actin, whereas uncharged or positively charged molecules retain significant activity.