T hyroxine-binding globulin (TBG) and partially desialylated or slow TBG (STBG) were purified from human serum by affinity chromatography. Purified TBG was identical to TBG present in serum by the criteria of electrophoretic mobility, affinity for thyroxine (T4), and heat-inactivation response. Purified STBG had slower electrophoretic mobility and lower affinity for T4. Both bound T4 in an equimolar ratio, were immunoprecipitable, and had similar inactivation t1/2 at 61 degrees C. TBG and STBG were iodinated by the chloramine-T-catalyzed reaction. An average of from 0.02 to 6 atoms I could be incorporated per molecule of the protein by adjusting the conditions of the reaction (time, protein and iodide concentrations). 125-I, 131-I, and 127-I were used. Iodination increased the anodal mobility of TBG but did not affect the reversible T4-binding, precipitation by antiserum, or the heat-inactivation properties. "Heavily" and "lightly" iodinated TBG had identical disappearance half-times from serum in the rabbit. 15 min after the intravenous administration of [131-I]-STBG and [125-I]TBG mixture to rats, more than 90% of the injected 131-I dose was in the liver, and the liver 131-I/125-I ratio was 32-fold that of serum. Selective uptake of STBG by the liver was also observed in the rabbit and in man. The serum [125-I]STBG/[131-I]TBG ratio declined from 1 to 0.2 in 10 min in the intact rabbit but remained unchanged for 1 h in the acutely hepatectomized animal. In the rabbit, t 1/2 was approximately 3 min for STBG and 0.8-3.4 days for TBG. The radioiodine derived from the iodinated proteins is partly excreted in bile but the bulk was precipitable with specific antibodies. Some isotope in the form of iodide appeared in blood and was excreted in the urine. Since radioiodinated TBG and STBG preserve their biologic and immunologic properties they are useful as tracer materials for metabolic studies. In rat, rabbit, and man STBG is rapidly cleared from serum by the liver. Conversion of TBG to STBG may be the limiting step in the regulation of TBG metabolism.