It has been suggested by other workers that prealbumin possesses 2 species of binding sites, one of which binds both thyroxine (T4) and tetraiodothyroacetic acid (TA4) and is inhibited by barbital, and the other of which binds only TA4 and is not affected by barbital. To test this hypothesis, 131I-labeled TA4 was synthesized from 131I-labeled T4 by rat kidney cortex incubated with serotonin. Binding of labeled TA4 and T4 in human serum was studied by the technique of filter paper electrophoresis in Tris-maleate buffer, pH 8.6. Relatively low concentrations of TA4 displaced tracer concentrations of T4 from prealbumin completely; in contrast, equimolar concentrations of T4 failed to displace tracer concentrations of TA4. Higher concentrations of T4, however, caused a progressive and ultimately an almost complete displacement of tracer concentrations of TA4 from prealbumin; this displacement conformed to a single linear function of the logarithm of the concentration of T4. In low concentrations, inhibitors of thyroxine-binding, such as barbital and 2,4-dinitrophenol, inhibited binding of T4 to prealbumin completely, but decreased the binding of TA4 only slightly. In higher concentrations, further inhibition of the binding of TA4 occurred, and, in the presence of 2,4-dinitrophenol, the molar concentration of TA4 displaced from prealbumin exceeded the maximum binding capacity for T4. The present data are inconsistent with the existence on prealbumin of 2 species of binding sites of distinctly different properties. They indicate, rather, that prealbumin possesses a single species of binding sites that binds TA4 more avidly than T4. (Endocrinology75: 917, 1964)